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https://f1000research.com/articles/12-51/v1
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12 Jan 23
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{
"type": "Review",
"title": "Application of transgenic zebrafish for investigating inflammatory responses to nanomaterials: Recommendations for new users",
"authors": [
"Helinor J Johnston",
"Suzanne L J Gillies",
"Rachel Verdon",
"Vicki Stone",
"Theodore Henry",
"Lang Tran",
"Carl Tucker",
"Adriano G Rossi",
"Charles R Tyler",
"Suzanne L J Gillies",
"Rachel Verdon",
"Vicki Stone",
"Theodore Henry",
"Lang Tran",
"Carl Tucker",
"Adriano G Rossi",
"Charles R Tyler"
],
"abstract": "Despite the increasing exploitation of nanomaterials (NMs) in an array of consumer products, there are uncertainties regarding their potential adverse impact on human health. Investigation of whether NMs activate a pro-inflammatory response is routinely used to assess their toxicity in in vitro and in vivo (rodent) studies. The use of zebrafish (Danio rerio) to investigate inflammatory responses to chemicals, pathogens and injury has increased considerably over recent years. Zebrafish have also been used to investigate the role of inflammation in disease pathogenesis and for drug discovery. Availability of transgenic strains which express fluorescent proteins in immune cells (e.g. macrophages and neutrophils) enables the visualization and quantification of immune cell accumulation in the target site(s) of interest. We therefore propose that transgenic zebrafish have great utility for screening the toxicity of NMs via investigation of inflammatory responses. Indeed, we have successfully used non-protected life stages of transgenic zebrafish with fluorescent neutrophils (Tg(mpx:EGFP114) to investigate inflammatory responses to NMs. The more widespread use of transgenic zebrafish in nanotoxicology could reduce the reliance placed on rodents and thereby enhance the implementation of the 3Rs principles. As zebrafish continue to grow in popularity it is timely to offer guidance to new users on their use. Here we will reflect on: exposure routes that can adopted to mimic human/rodent exposure, what transgenic strains and life stages are best suited to investigate inflammatory responses, selection criteria for zebrafish embryos/larvae, the inclusion of appropriate controls, the importance of dose selection and sample size, and how the (inflammatory) response can be quantified. It is hoped that our recommendations will support the development of standard protocols that can be used to assess whether NMs activate inflammatory responses. Importantly, the themes discussed are not restricted to NMs but relevant also to zebrafish application in ecotoxicology or human health focused studies.",
"keywords": [
"nanomaterial",
"nanotoxicology",
"zebrafish",
"neutrophil",
"inflammation"
],
"content": "\n\n\n\nScientific benefits\n\n\n\n• Non-protected life stages of transgenic zebrafish can be used to screen the toxicity of nanomaterials (NMs) via investigation of inflammatory responses.\n\n• Zebrafish could provide better predictions of the impact of substances on human health than rodents\n\n3Rs benefits\n\n\n\n• The use of non-protected life stages of zebrafish could reduce and/or replace the use of rodents in nanotoxicology\n\nPractical benefits\n\n\n\n• The inflammatory response to NMs can be monitored in the same zebrafish over time, a major advantage over rodents\n\n• The use of zebrafish can make testing cheaper and quicker\n\nCurrent application\n\n\n\n• Zebrafish can be exploited to assess the adverse impact of NMs on human health via investigation of inflammatory responses.\n\nPotential application\n\n\n\n• A wider panel of NMs should be assessed in the future\n\n• Different administration routes should be considered to better mimic human exposure to NMs\n\n• Different transgenic strains should be explored to further probe the mechanism of NM toxicity\n\n• The model is not restricted to nanotoxicology and should be exploited more widely by other disciplines\n\n\nIntroduction\n\nToxicity testing has traditionally relied on the use of rodents when assessing the potential impact of substances on human health, often following test guidelines from the Organisation for Economic Co-operation and Development (OECD). Use of alternative (non-rodent) models is increasing to promote alignment with the 3Rs principles of reduction, refinement and replacement of animal use in scientific research, to make toxicology testing more ethical, quicker and often cheaper. The concern that rodents do not always provide a good prediction of human responses further illustrates the need to find alternative models for toxicology testing (Hartung, 2009).\n\nZebrafish (Danio rerio) are a highly versatile vertebrate model used widely for biomedical and (eco) toxicology research (Lieschke and Currie 2007). A prominent use of zebrafish is in the hazard assessment of chemicals, and in particular for ecotoxicity testing using the validated Fish Embryo Acute toxicity (FET) test which assesses the impact of substances on zebrafish embryo development (OECD Test Number 236, 2013, Hill et al., 2005). Over recent years the use of zebrafish for human health related research has expanded. More specifically, zebrafish have been used to study the pathogenesis of many diseases such as cancer (e.g., Hason and Bartůněk 2019, Letrado et al., 2018, Stewart et al., 2014, Lu et al., 2015), cardiovascular disease (e.g. Bournele and Beis 2016, Taylor et al., 2019), neurological diseases (e.g., Fontana et al., 2018, Ünal and Emekli-Alturfan 2019), infectious diseases (e.g., Gomes and Mostowy 2020, Torraca and Mostowy 2018), and inflammatory diseases that affect different organs such as the lungs, intestine and liver (e.g. Renshaw et al., 2007, Brugman, 2016, Zhang et al., 2016, Hanyang et al., 2017, Shwartz et al., 2019). In addition, zebrafish are increasingly used to accelerate drug discovery by screening the bioactivity and toxicity of new compounds (Vliegenthart et al., 2014a, MacRae and Peterson 2015, Keßler, Rottbauer and Just 2015, Letrado et al., 2018, Bournele and Beis 2016, Hoodless et al., 2016, Yoganantharjah and Gibert 2017, Stewart et al., 2014, Hill et al., 2005, Horzmann and Freeman 2018).\n\nThere are many advantages of using zebrafish over rodents for toxicology testing when investigating the potential impact that substances have on human health. Firstly, studies that have employed zebrafish for toxicology testing have shown good correlation with human data (Nadanaciva et al., 2013, Sukardi et al., 2011, Sipes et al., 2011, Tal et al., 2020). For example, excellent concordance (100%) between zebrafish and humans was observed when assessing drug induced hepatotoxicity (He et al., 2013). Similarly, zebrafish had a 100% prediction success rate for cardiotoxic drugs in humans (Zhu et al., 2014). In addition, a meta-analysis for >600 chemicals indicated that zebrafish could be used as an initial screen to prioritise chemicals for rodent testing and as a potential replacement for mammalian testing (Ducharme et al., 2015). Of benefit is that biomarkers for toxic responses can often be measured across different species; for example, the liver-enriched microRNA (miRNA) miR-122 is a highly conserved biomarker for hepatoxicity which can be assessed in zebrafish and could be used to predict hepatotoxicity of substances in humans (Vliegenthart et al., 2014b). Other advantages of using zebrafish over rodents include their small size, relative ease and cost of maintenance compared with rodents, high fecundity, availability of genetically manipulated (transgenic) strains, embryo/larval transparency (e.g. which facilitates direct in vivo visualization of developmental processes, and organ/tissue structure and function), and their amenability to high throughput testing. Furthermore, a major advantage associated with the use of zebrafish in toxicology (and other disciplines) is their potential to offer many 3Rs benefits, which are discussed in more detail later. In the EU, early life stages of zebrafish, prior to exogenous (independent) feeding at 120 hours/5 days post fertilisation (hpf or dpf)), are not protected by law and as such do not require permission from responsible authorities to be used in scientific research (EU Directive 2010/63/EU). However, as adult fish are required to obtain a supply of zebrafish embryos/larvae, then the adult fish would need to be housed in approved facilities.\n\n\nTransgenic zebrafish\n\nBoth wild type and transgenic strains of zebrafish have been used in existing studies that have a human health focus. The optical transparency of zebrafish embryos and larvae combined with the expression of fluorescent proteins in transgenic zebrafish lines allows real time imaging of cellular processes, organelles, cells and tissues (He et al., 2014, Lee et al., 2015, Bai and Tang 2020, Choe et al., 2021). Transgenic zebrafish which express fluorescent proteins have been developed for visualisation of a diverse range of targets including but not limited to immune cells (Renshaw et al., 2006, Ellett et al., 2011), neurones (Park et al., 2000, Winter et al., 2021), blood vessels (Gao et al., 2016), the liver (Zhang et al., 2014), pancreas (Huang et al., 2001), kidney (Perner et al., 2007), mitochondria (Kim et al., 2018, Zhou et al., 2018), Golgi apparatus (George et al., 2014), oestrogen receptors (Lee et al., 2012, Green et al., 2018), ovaries (Akhter et al., 2016), markers of oxidative stress (Kusik et al., 2008, Mourabit et al., 2019), and intracellular signalling pathways (e.g. NfkB (Kanther et al., 2011)) (reviewed by e.g. Choe et al., 2021, Lee et al., 2012).\n\nKnockout strains of zebrafish are also available which allow the role of specific genes to be probed in studies that focus on investigating disease pathogenesis, toxicity and drug discovery. Of particular relevance to this paper is the use of knockout strains for investigating the role of different immune cell types in inflammatory responses; however, the use of knockout strains is not the focus of this paper. Examples include where chemokine (CXC) ligand-8 (CXCL8) knockouts have been used to investigate the role of CXCL8 in neutrophil recruitment (de Oliveira et al., 2013), and the use of knockout strains to demonstrate that mitochondria are important to neutrophil motility in vivo (Zhou et al., 2018). Approaches available to deplete functional macrophages and neutrophils from zebrafish may also be of interest and have been reviewed by Rosowski (2020).\n\n\nZebrafish and inflammation\n\nComponents of the immune system of zebrafish are similar to that of humans, with the key cell lineages present including monocytes, tissue macrophages, granulocytes (e.g., neutrophils, eosinophils) and lymphocytes (Traver et al., 2003, Meeker and Trede, 2008, Novoa and Figueras, 2012). The innate immune system is functional within 48 hpf, with the adaptive system developing after ~4 weeks (Renshaw et al., 2006, Trede et al., 2004, Novoa and Figueras, 2012). The ability to focus on the innate inflammatory response alone is often considered an additional benefit of using early life stages of zebrafish in biomedical research.\n\nOver the last decade, the use of zebrafish as an alternative to rodents for investigating inflammatory responses to various stimuli (e.g. chemicals, pharmaceuticals, pathogens and injury) has increased considerably. Zebrafish have also been used to investigate the contribution of inflammation to disease pathogenesis. A PubMed search in June 2022 (using the search terms zebrafish AND inflammation) revealed a total of 1697 published papers for the years between 2000 and 2022. The increasing interest in the use of zebrafish to investigate inflammatory responses is further illustrated by the knowledge that in 2000 there was only one published paper in this area, whereas in 2021 there were 308 publications (with >70% of the existing papers published in the last five years).\n\nIt is often desirable to monitor inflammatory responses over time in animal models as this allows capture of the initiation, peak, and resolution (or lack thereof) of inflammation. Methods have been developed to visualize immune cell migration in rodents in real time using intravital microscopy (Khandoga et al., 2009, Xu, Lei and Liu, 2011, reviewed by De Filippo and Rankin, 2020). However, this approach is not routinely used, requires specialized equipment, is not applicable to all tissues/organs and typically assesses responses over short time frames (several hours). Instead, it is common to include several groups of rodents in each in vivo study to investigate inflammatory responses that are activated at different time points, with the accumulation of immune cells typically assessed by performing differential cell counts (e.g. from bronchoalveolar lavage (BAL) or blood samples) or by visualizing the inflammatory response using histology.\n\nHistological examination has also been used to visualise inflammatory responses in wild type zebrafish at various life stages (e.g., Yang et al., 2014). However, the availability of transgenic zebrafish which express fluorescent proteins in specific immune cell types allows the direct visualization and quantification of immune cell accumulation in specific locations as well as the whole organism (Figure 1). Importantly, pigmentation starts developing in zebrafish from 1 dpf, and although is not fully developed until 14 dpf (Rawls et al., 2001) this can make it challenging to visualize fluorescent cells as zebrafish age. For this reason, the majority of existing studies typically assess inflammatory responses in early life stages. However, there are several strategies that can be used to improve the transparency of zebrafish larvae, such as the use of (toxic) chemicals to stop the creation of pigment (e.g., 1-phenyl-2-thiourea, PTU) or the use of mutant strains which lack skin pigmentation (e.g., casper strains of zebrafish) (Antinucci and Hindges 2016). To date, the majority of existing research has focused on assessing neutrophil and macrophage responses in zebrafish, using transgenic lines with fluorescent neutrophils (e.g., Tg (mpx: GFP)), fluorescent macrophages (e.g., Tg (mpeg1):GFP) (Renshaw et al., 2006, Ellett et al., 2011) or both (e.g., Tg (mpx: GFP/mpeg1:mCherry)) (Ellett et al., 2011). It is most common for images of zebrafish to be captured at specific time points using fluorescent microscopy to visualize and quantify the inflammatory response over time, however imaging can also be performed in real-time. As inflammatory responses can be monitored in the same organism over time this can lead to a reduction in animal use, which is a major benefit of using transgenic zebrafish over rodents. More specifically, studies using (non-protected life stages of) zebrafish commonly investigate inflammatory responses to specific stimuli (e.g., injury, injection of pathogens) at multiple time points (ranging from one to seven, with an average of three) in the same organism, (Tables 1 and 2). As a comparison, a PubMed search in Oct 2022 (search terms: nanomaterial OR nanoparticle AND lung AND rat or mouse AND inflammation AND neutrophil) revealed that rodent studies used an average of three time points (range of one to six) to quantify pulmonary inflammatory responses to NMs. Therefore, each rodent study investigating inflammatory responses to NMs required the use of multiple groups of animals to gather information at each time point of interest, with separate control groups required for each time point. Zebrafish allow the inflammatory response to be monitored in the same organism over time to maximise the amount of information obtained from each organism, perform a more comprehensive assessment of the dynamics of the inflammatory response (via the use of multiple time points) and reduce the number of treatment groups (and therefore number of animals) required (compared to rodents).\n\nImage taken by Dr Suzanne Gillies using a fluorescent Leica M205 FCA stereomicroscope. Scale bar is 250 μm.\n\nTransgenic zebrafish with fluorescent immune cells have been used to investigate inflammatory responses to various stimuli including, for example, lipopolysaccharide (LPS) (e.g., Yang et al., 2014, Zhang et al., 2016), neutrophil chemoattractants (e.g., chemokine (CXC) ligand-8 (CXCL-8), N-Formyl-Met-Leu-Phe (fMLF), Leukotriene B4 (LTB4) (Elks et al. 2011, Bischel et al. 2013, de Oliveira et al., 2013)), micro-organisms (e.g. Nguyen-Chi et al., 2014, Benard et al., 2012, Jim et al., 2016), injury (tail fin wound; (e.g., Renshaw et al., 2006, Ellett et al., 2011, Hoodless et al., 2016, Miskolci et al., 2019, Table 1), burns (thermal injury) (Miskolci et al., 2019) and cardiac injury (e.g., Kaveh et al., 2020)). In addition, transgenic zebrafish have been used to investigate the contribution of inflammation to disease pathogenesis (e.g. pulmonary disease (Zhang et al., 2016), cardiovascular disease (Taylor et al., 2019, Kaveh et al., 2020), intestinal disease (Brugman, 2016), liver disease (Shwartz et al., 2019), cancer (Yang et al., 2019, Wrighton et al. 2019)), and tissue repair/regeneration (Renshaw et al., 2006, Li et al., 2012, Petrie et al., 2014). Transgenic zebrafish have also been used for drug discovery to identify novel anti-inflammatory compounds (e.g., Hoodless et al., 2016, Lucas et al. 2013, Yang et al., 2014, Rahman et al., 2019, Xie et al., 2021).\n\n\nNanomaterials and inflammation\n\nNanomaterials (NMs) are defined as having at least one dimension that is 1–100 nm in diameter (European Commission, 2011). The properties exhibited by materials at the nanoscale can be drastically different to those observed in larger forms of the same material. This has promoted the incorporation of NMs into a wide variety of consumer products (Vance et al., 2015) with the use of NMs now spanning a very wide range of sectors (e.g., pharmaceuticals, cosmetics, textiles, food, electronics, automotive, construction, agriculture, and pigments/inks). Although the prevalence of NMs in the marketplace continues to increase, there are uncertainties regarding their potential adverse impact on human health. Thus, a thorough assessment of NM toxicity needs to be conducted in parallel to exploring their benefits to ensure that nanotechnology develops in a safe, responsible and sustainable manner. NMs are an extremely diverse group of materials and due to the large number of NMs for which safety needs to be assessed there is a desire to move away from rodent testing to improve alignment of nanotoxicology research with the 3Rs principles (Burden et al., 2017, Johnston et al., 2018).\n\nInvestigation of whether NMs activate inflammatory responses is routinely used to assess their toxicity both in vitro and in vivo (reviewed by Johnston et al., 2018). In vitro studies have used a variety of cell types from different target sites (e.g., immune system, lung, liver, intestine, skin) to assess whether NMs can stimulate cytokine production as an indicator of their potential to activate inflammatory responses. Such studies have shown that, depending on their physico-chemical properties, NMs can enhance the production of neutrophil (e.g., interleukin (IL)-8, growth-regulated oncogene alpha (GROα), macrophage inflammatory protein (MIP)-2) and macrophage (e.g., macrophage chemotactic protein (MCP)-1) chemoattractants as well as other pro-inflammatory cytokines (e.g., tumour necrosis factor (TNF)-α, IL-1β) (e.g., Verdon et al., 2021, Zhang and Monteiro-Riviere, 2019, Dankers et al., 2018, Johnston et al., 2015, Braakhuis et al., 2016, Wiemann et al., 2016, Sahu et al., 2014, Kermanizadeh et al., 2013, Farcal et al., 2013, Goncalves et al., 2010, Brown et al., 2004). Investigation of the activation of immune cells in vitro (typically macrophages and neutrophils) following exposure to NMs has also been evaluated via investigation of a range of cell responses (e.g., cytotoxicity, cytokine production, activation of respiratory burst, phagocytic function, cell motility, and neutrophil extracellular trap formation (NETosis)) (e.g., Goncalves et al., 2010, Sahu et al., 2014, Wiemann et al., 2016, Johnston et al., 2015, Verdon et al., 2021). Whilst cell based, in vitro assays often offer a quick, cheap and high-throughput method of assessing the inflammatory effects of NMs, they are not able to accurately reflect the in vivo situation as they cannot mimic the complexity of interactions that occur between immune and non-immune cells during inflammatory responses in humans. Furthermore, whilst the production of both pro- and anti-inflammatory mediators can be monitored over time using in vitro models, it is challenging to investigate the resolution of inflammatory responses outside an intact organism.\n\nRodent studies have therefore been used to investigate the dynamics of inflammatory responses that are activated by NMs, and to date the majority of these have focused on pulmonary inflammatory responses. The activation of an inflammatory response (i.e., infiltration of leukocytes into the target site) and failure of that response to resolve in vivo is indicative that a NM may be toxic. Studies have consistently demonstrated that NMs (depending on their physico-chemical properties) can activate pro-inflammatory responses that are often characterised by an accumulation of neutrophils (e.g., Brown et al., 2001, Poland et al., 2008, Ma-Hock et al. 2009, Srinivas et al., 2012, Murphy et al., 2013, Bonner et al., 2013, Kim et al., 2016, Landsiedel et al. 2014, Gosens et al., 2015, Halappanavar et al., 2019). Macrophages also play a key role in the response of organs/tissues (e.g., lungs) to NMs, and the clearance of NMs by resident macrophages is likely to promote an inflammatory response (e.g., Ogawara et al., 1999, Sadauskas et al., 2007, Semmler-Behnke et al., 2008, Geiser et al., 2008, Roberts et al. 2013, Anderson et al., 2015, Gustafson et al., 2015).\n\n\nZebrafish and nanotoxicology\n\nTo date, the majority of nanotoxicology studies that have employed zebrafish have focused on investigating the toxicity of NMs to the environment. Such studies have assessed NM hazards via evaluation of the impact of NMs on zebrafish survival, growth, development, reproduction, hatching rate, and teratogenicity following aqueous exposure (e.g., Lee et al., 2007, Usenko et al., 2008, Asharani et al., 2008, Zhu et al., 2009, Fent et al., 2010, Massarsky et al., 2013, Duan et al., 2013, Pavagadhi et al., 2014, Wehmas et al., 2015, Ganesan et al., 2016, Pecoraro et al., 2017, Vranic et al., 2019, Aksakal and Ciltas, 2019). It is most common for such studies to use wild type zebrafish.\n\nTransgenic zebrafish have been used to a more limited extent to assess the potential detrimental impact of NMs on human health via investigation of responses such as inflammation (e.g., using the Tg (mpx: EGFP)i114 strain which expresses fluorescent neutrophils), cardiovascular toxicity (e.g., using the Tg (fli-1:EGFP) strain which expresses EGFP-labelled vascular endothelial markers, fli-1), and neural toxicity (e.g., using the Tg (HuC-GFP) strain that expresses GFP in the central nervous system) (e.g. Bar-Ilan et al., 2012, Duan et al., 2016, Gao et al., 2016, Vranic et al., 2019, Gu et al., 2021, Gillies et al., 2022).\n\nInflammatory responses have been evaluated in wild type zebrafish following aqueous exposure through histological examination of the gills (Filho et al., 2014) and skin (McLeish et al. 2010). In addition, Brun et al., (2018) demonstrated that the expression of pro-inflammatory genes and infiltration of neutrophils increased in the skin and intestine of transgenic zebrafish embryos (with fluorescent TNFα, IL-1β, neutrophils or macrophages) following aqueous exposure to copper and polystyrene NMs. A small number of published studies have investigated inflammatory responses to NMs following microinjection into various body sites of transgenic zebrafish larvae with fluorescent neutrophils (e.g., Sharif et al., 2012, Zhang et al., 2016, Brun et al., 2018, Duan et al., 2016, 2018, Gillies et al., 2022). Only one study was identified in the literature that has investigated inflammatory responses to NMs in (injured) transgenic zebrafish following aqueous exposure (Gillies et al., 2022). Therefore, whilst transgenic zebrafish have been used to assess NM toxicity, these studies have assessed a very limited number of NMs, administration routes, target sites, time points, zebrafish strains and life stages. There is an opportunity now to apply transgenic zebrafish to a far greater extent for assessing the potential adverse impacts of NMs on human health. We therefore propose that the more widespread screening of NM toxicity via investigation of inflammatory responses in transgenic zebrafish could make nanotoxicology testing more ethical, faster, cheaper and potentially more predictive.\n\nThe use of transgenic zebrafish in nanotoxicology studies to investigate impacts of NMs on human health is in its infancy, however results from existing studies are very promising. For example, as part of an NC3Rs funded project, we assessed the suitability of using transgenic zebrafish to investigate whether NMs could stimulate an inflammatory response in a novel approach to assess their toxicity (Gillies et al., 2022). We selected silver (Ag) NMs, and zinc oxide (ZnO) NMs as in vivo rodent data on their capacity to elicit a pulmonary inflammatory response already existed (e.g., Gosens et al., 2015, Li et al., 1996, Landsiedel et al., 2014). Furthermore, there is extensive evidence that these NMs stimulate cytokine production by various cells types in vitro (e.g. Gaiser et al.,2013, Kermanizadeh et al., 2013, Farcal et al., 2015, Johnston et al., 2015, Verdon et al., 2021). Human exposure to these NMs is also expected due to the use of these NMs in a range of consumer products. We investigated the dynamics of the neutrophil responses that were activated by NMs following i) aqueous exposure of injured transgenic zebrafish larvae (with fluorescent neutrophils) or ii) microinjection into the otic vesicle of transgenic zebrafish larvae (Gillies et al., 2022). Only non-protected life stages of zebrafish were used in our studies to support the implementation of the 3Rs principles. We demonstrated that transgenic zebrafish can be effectively employed to assess the inflammatory effects of NMs (Gillies et al., 2022) and would therefore encourage the more widespread use of the model by the nanotoxicology community.\n\nAs there is increasing interest in the exploitation of zebrafish to assess inflammatory responses in nanotoxicology and other disciplines, we believe it is timely to present recommendations to new users of the model. There is no standard protocol available to assess inflammatory responses to NMs (or other substances/pathogens) using transgenic zebrafish and within the existing literature the methodology used in different studies to investigate inflammatory responses in transgenic zebrafish is varied (e.g. with respect to the zebrafish strain, life stage, administration route, sample size and time points used as well as the approach used to quantify the inflammatory response) which can make it challenging for new users of the model to design their experiments. Here we therefore provide guidance to new users on how transgenic zebrafish can be used to investigate inflammatory responses to NMs. We will also identify gaps in knowledge to help prioritise future research activities. Importantly, the themes we discuss are not restricted to NMs and are relevant to other substances and pathogens. Furthermore, we focus on assessment of impacts relaying more to human health but many of the topics covered are also relevant to ecotoxicology studies.\n\n\nSelection of exposure route/target site\n\nIt is important to consider how zebrafish should be exposed to NMs if designing work to infer for effects for exposure routes that are relevant to humans. Assessment of NM toxicity following pulmonary exposure (e.g., inhalation, intratracheal instillation) has dominated existing in vivo rodent studies (Stone et al., 2016). Relatively fewer rodent studies have exposed rodents to NMs orally, via the skin or intravenous injection (Stone et al., 2016); however, exposure of humans to NMs via these routes is expected in occupational, environmental, and consumer settings.\n\nThe most convenient and popular way of exposing zebrafish to substances (e.g., NMs) is via water (Sipes et al., 2011), but this may not always be the most appropriate route when considering relevance for human health related research. Whilst microinjection is more technically challenging and requires access to specialised equipment, it allows for the administration of NMs into specific sites and subsequent investigation of inflammatory responses locally at the injection site, as well as systemically. Indeed, inflammatory responses to a range of different stimuli (such as LPS, neutrophil chemoattractants and pathogens (e.g., bacteria, fungi)) have been investigated following their microinjection into several sites in zebrafish embryos/larvae including the otic vesicle, swimbladder, hindbrain ventricle, notochord, and caudal vein (e.g., Gratacap et al., 2014, Nguyen-Chi et al., 2014, Benard et al., 2012, Jim et al., 2016, Harvie and Huttenlocher 2015, de Oliveira et al., 2013) (Figure 2). Recommendations are provided below regarding how zebrafish can be exposed to NMs when assessing responses following pulmonary, oral, and dermal exposure as well as intravenous injection.\n\nFigure created in BioRender.com.\n\n\n‘Pulmonary’ exposure\n\nWe have outlined below how zebrafish may be exposed to NMs via water or microinjection to mimic pulmonary (respiratory) exposure.\n\nInterestingly, zebrafish have been used to assess the pathogenesis of pulmonary diseases, despite their lack of a mammalian-like respiratory system (Renshaw et al. 2007, López Hernández et al., 2015, Progatzky et al., 2016, Zhang et al., 2016). Fish respire via gills, and these tissues perform an equivalent gas-exchange function to mammalian lungs (Progatzky et al., 2016), as well as playing a key role in other critical functions such as ion and pH regulation and the excretion of nitrogeneous waste (Evans et al., 2005, Dymowska et al., 2012). The zebrafish gill is the primary site of gas exchange and has a complex vasculature that is covered by an epithelium which acts as a barrier between the blood and external environment (Evans et al., 2005). Within the structure of the gills there are several different cell types including epithelial cells, ionocytes (which control ion transport), inflammatory cells (e.g., lymphocytes and neutrophils), mucus-producing (goblet) cells, and skeletal muscle cells. Unidirectional flow of water over the gills (as opposed to tidal flow of air in lungs of mammals), production of mucus, and coughing during ventilatory movements combine to enable fish to dislodge and remove materials (e.g., particulates, including NMs) from gill surfaces.\n\nThere is evidence that NMs can accumulate in the gills of fish following aqueous exposure (Johnston et al., 2010, Zhao et al., 2011, Bar-Ilan et al., 2013, Bacchetta et al., 2016, Skjolding et al., 2017). For example, it has been shown that the gills of zebrafish are the primary site of copper oxide (CuO) NM accumulation and toxicity following aqueous exposure (Griffitt et al., 2007). Furthermore, Tang et al., (2019) demonstrated that TiO2 NMs could cause oxidative damage to the adult zebrafish gill following aqueous exposure. Interestingly, Filho et al. (2014) showed (using histological examination) that carbon nanotubes (CNTs) stimulated an inflammatory response in the adult zebrafish gill following aqueous exposure. Given the above, inflammatory responses in the gills of zebrafish in response to NM exposure via water could provide a potential surrogate for assessing pulmonary inflammatory responses in rodents. However, zebrafish gills do not start to develop until 3 dpf, and they are not fully developed and functional until >14 dpf (Rombough, 2002). Therefore, protected life stages of zebrafish would likely have to be used for assessing NM mediated inflammatory responses at this target site, with the associated ethical implications and animal licencing requirements. Using protected life stages of zebrafish, however, may still have 3Rs benefits as it allows for a reduction in the use of mammals in toxicology testing and it may be possible to monitor responses in the same organism over time due to the transparency of early life stages of zebrafish, which will reduce the number of animals (fish) used in each experiment. However, this will depend on whether the increased pigmentation of zebrafish which increases as they age obscures the visualization of fluorescent immune cells in transgenic zebrafish (see above).\n\nThe skin is the main site of gas exchange in zebrafish embryo/larvae until the gills are developed, and it has been suggested that the skin epithelium has a similar function to the alveolar epithelium in humans (Rong et al., 2021). The skin epithelia could therefore have potential for use to assess the respiratory toxicity of NMs following aqueous exposure in non-protected life stages of zebrafish (Schwerte, 2010).\n\nAqueous exposure of zebrafish to NMs is not technically challenging (e.g., Gillies et al., 2022), does not require specialised equipment and has the added benefit that repeated exposures can be performed with the same animals. Thus, transgenic zebrafish with fluorescent immune cells could potentially be used to investigate inflammatory responses to NMs at the gills or skin as an alternative to assessing inflammatory responses following pulmonary exposure in rodents.\n\nThe swim bladder is a gas filled sac which regulates buoyancy in zebrafish and has anatomical similarities in its cellular make up to the alveoli of the human lung (Finney et al., 2006). Furthermore, the swim bladder shares a common evolutionary origin with the mammalian lung (Zheng et al., 2011). The swim bladder has three distinct layers of tissue; a single layer of epithelial cells covering a mesenchymal layer which is then surrounded by an outer mesothelial layer (Yue et al., 2015). The epithelium is lined with a thin fluid (mucosal) layer containing surfactants (as in the mammalian lung) (Prem et al., 2000). Zhang et al., (2016) observed that silica NMs, LPS, Polyinosinic:polycytidylic acid and neutrophil chemoattractants (IL-8 and MIP-2) stimulated an infiltration of neutrophils into the swim bladder of transgenic zebrafish larvae following microinjection. No other studies were identified in the literature that have injected NMs into the swim bladder of zebrafish to investigate their toxicity. Investigation of inflammatory responses in the swim bladder of transgenic zebrafish larvae (with fluorescent immune cells) has been used in previous studies to investigate lung injury (Zhang et al., 2016, Lee et al., 2019), as well as host-pathogen interactions (e.g. the fungal pathogen Candida albicans) (Gratacap et al., 2014). Thus, microinjection of NMs into the swim bladder could also be an appropriate method to investigate pulmonary responses to NMs. Gratacap et al., (2014) provide guidance on how to perform swim bladder microinjections for fungal pathogens. However, the swim bladder is not fully developed until 4–5 dpf and so investigation of NM mediated inflammatory responses in this location may require the use of protected life stages of zebrafish (depending on how long inflammatory responses were monitored for) and thus there may be more ethical implications of using this exposure route and again permission will be required from responsible authorities to perform this type of research.\n\n\nOral exposure\n\nThe toxicity of NMs following oral exposure has been investigated in several rodent studies (e.g., de Jong et al., 2019, Park et al., 2010, Heo et al., 2020) and thus it is appropriate to consider how zebrafish can be used to investigate the fate and toxicity of ingested NMs. Exposure of fish to NMs via water can lead to NM accumulation in the gastrointestinal tract (GIT) via drinking (e.g., Lin et al., 2014, Zhao et al., 2011, van Pomeron et al., 2017). Alternatively, NMs can be incorporated into the diet of fish (e.g., Geffroy et al., 2012, Merrifield et al., 2013). Indeed, localisation of NMs in the intestine has been observed in zebrafish exposed to NMs via the diet (Skjolding et al., 2017). Ingestion of food containing Ag and Cu NMs by adult zebrafish for 14 days has been observed to disrupt the gut microflora (Merrifield et al., 2013). Furthermore, Brun et al. (2018) observed that intestinal tissue pro-inflammatory cytokine expression was increased, and that there was an increase in macrophage and neutrophil accumulation in the intestine following aqueous exposure to Cu NMs. Zebrafish have also been successfully used to investigate the role of inflammation in intestinal diseases (reviewed by Brugman, 2016). The gastro-intestinal tract opens after 3 dpf, however zebrafish do not feed independently until ~5 dpf, up until which time they are reliant on a yolk sac reserve. Consequently, protected life stages of zebrafish are required to investigate the toxicity of ingested NMs. Given the absence of studies which have investigated inflammatory responses to NMs in the intestine following ingestion this would seem to be an area of research that should be prioritised for the application of transgenic zebrafish in the future.\n\n\nDermal exposure\n\nThe majority of existing studies have used in vitro approaches to assess the dermal toxicity of NMs (e.g., Monteiro-Riviere et al. 2005, Rouse et al. 2008, Sayes et al. 2006, Shvedova et al., 2003, Zhang et al., 2008, Connolly et al., 2019, reviewed by Crosera et al., 2009) and far fewer studies have investigated dermal toxicity of NMs in vivo (e.g. Samberg et al., 2010). Cytokine production by in vitro skin models of varied complexity (e.g., monocultures of skin cells (e.g., keratinocytes), complex 3D models of the skin (e.g., EpiDerm, and human/animal tissue) can be used to investigate whether NMs activate an inflammatory response. Several in chemico and in vitro OECD test guidelines (that recommend the use of more advanced in vitro models) are available that can be used to screen the dermal toxicity of substances (e.g. via assessment of irritation and sensitisation) (e.g. OECD Test Nos 432, 439, 442C, 442E). Indeed, the dermal toxicity of NMs has already been screened using OECD methods (e.g., Park et al., 2011, Bezerra et al., 2021). Complex, physiologically relevant 3D skin models (with multiple cell layers) have also been used to assess NM toxicity in vitro as an alternative to animal testing and most commonly to assess skin irritation (loss of cell viability) (e.g., Park et al., 2011, Choi et al., 2014, Kim et al., 2016, Miyani and Hughes 2017, Franková et al., 2018, reviewed by Sanches et al., 2020). It is also common for researchers to assess the response of (a single layer of) keratinocyte cell lines when investigating the dermal toxicity of NMs in vitro. For example, cytokine production by keratinocytes has been used to screen the dermal toxicity of NMs in vitro (Samberg et al., 2010, Saathoff et al., 2011, Jeong et al., 2013, Murray et al., 2013, Galandáková et al., 2016). However, there is evidence that more simple, less physiologically relevant models are more sensitive to NM toxicity (Chen et al., 2019). Whilst 3D in vitro models of the skin are available and have been used as an alternative to animal (rodent) testing, they lack the ability to assess the migration and accumulation of immune cells in the skin following NM exposure. Assessment of such responses is desirable to assess the contribution of inflammation to the dermal toxicity of NMs. Non protected life stages of zebrafish may therefore offer an alternative model to assess the dermal toxicity of NMs in vivo and more specifically to investigate the activation of inflammatory responses within the skin. Indeed, there is evidence that NMs accumulate in the skin of zebrafish following aqueous exposure (e.g., Zhao et al., 2011, van Pomeron et al., 2017), and that NMs can exhibit toxicity to the skin of zebrafish. For example, Brun et al., (2018) found that CuO NMs increased the expression of the pro-inflammatory cytokine IL-1β and the accumulation of immune cells (such as macrophages and neutrophils) in the skin of transgenic zebrafish embryos, following aqueous exposure. In addition, McLeish et al., (2010) demonstrated (using histology) that NMs caused skin damage in zebrafish larvae, and induced leukocyte infiltration into the epidermis following aqueous exposure. More recently, exposure of zebrafish to Cu and Ag NMs has been shown to cause oxidative stress in various types of ionocytes which are cells that are present in the skin and responsible for mediating ion exchange in zebrafish embryo/larvae (Takesono et al., manuscript in preparation).\n\nZebrafish embryos are protected by a chorion up to 3 dpf that acts as a barrier and limits the interaction of NMs (and other substances) with the embryo (Brun et al., 2018, van Pomeron et al., 2017, Fent et al., 2010). This has major implications when considering the life stage that is used to assess the dermal impacts of NMs on zebrafish, and it may be preferable to use zebrafish embryos that have hatched from the chorion (Brun et al., 2018). Alternatively, embryos can be dechorionated manually or by using chemicals/enzymes to allow skin exposure at earlier time points, although this can be time consuming. Following hatching, the main route of exposure in non-protected life stages will be via the dermal route, as the gills are not fully developed and the embryo cannot independently feed at this time (Sipes et al., 2011). Due to the limited number of studies which have investigated inflammatory responses to NMs in the skin of transgenic zebrafish, this is a knowledge gap that needs researching.\n\nWhen assessing inflammatory responses in zebrafish embryo/larvae following dermal exposure it may be of interest to investigate the responses of neuromasts. Neuromasts are mechanosensory cells that comprise the lateral line system but are also found more widely across the body surfaces of zebrafish (Froehlicher et al., 2009). It has been suggested that investigation of the impact of chemicals on neuromast development and function can be used to assess toxicity (reviewed by Froehlicher et al., 2009). Previously, neuromast cell death has been assessed to investigate the toxicity of metals, such as copper, and pharmaceuticals, (e.g., Mourabit et al., 2019). In addition, McNeil et al., (2014) demonstrated that exposure of zebrafish embryos to Cu NMs reduced the number of functional lateral line neuromasts which affected fish behaviour. As only a few studies have assessed the toxicity of NMs to neuromasts this should be explored further in the future to address knowledge gaps.\n\n\nIntravenous exposure\n\nIntravenous injection provides a way of directly introducing NMs into blood at a specific dose and can be used to identify the implications of NMs on health if they become systemically available following exposure. Exposure of rodents via intravenous injection is commonly used to investigate the fate and toxicity of NMs that may be used in a clinical setting (e.g., nanomedicines) as they may be administered via this route. Several rodent studies have investigated NM biodistribution and toxicity following intravenous injection (e.g., Semmler-Behnke et al., 2008, Fujihara et al., 2015, Disdier et al., 2015). It is therefore prudent to reflect on how to administer NMs to zebrafish to mimic intravenous injection. When investigating host-pathogen interactions in zebrafish larvae, pathogens have frequently been injected into the caudal vein or Duct of Cuvier (a circulation channel located on the yolk sac, and responsible for connecting the heart to the trunk vasculature) to cause systemic blood infections (Benard et al., 2012, Harvie and Huttenlocher, 2015). In addition, the cardiotoxicity of silica NMs following their injection into the Duct of Cuvier has been investigated previously, with cardiac inflammation (neutrophil accumulation) observed following NM exposure (Duan et al., 2016). As investigation of whether NMs can activate systemic inflammatory responses following intravenous injection into non-protected life stages of transgenic zebrafish larvae has been neglected to date it is suggested that this knowledge gap is addressed in future studies.\n\nThe yolk sac contains a supply of nutrients required by the developing zebrafish until exogenous feeding beings at 5 dpf (Quinlivan and Farber 2017). Microinjection of pathogens and substances into the yolk sac of zebrafish has been used as a systemic infection/exposure model (Benard et al., 2012, Yang et al., 2014, Jim et al., 2016, Xie et al., 2021). The distribution of fluorescent polystyrene NMs following microinjection into the yolk sac has also been investigated in zebrafish embryos (Zhang et al., 2020); however no other published studies were identified in which the fate or toxicity of NMs following injection into the yolk sac were investigated. Administration of NMs via the yolk sac could be an effective way to introduce NMs such that they become systemically available and therefore this route of exposure could be used as a surrogate to intravenous injection, but more studies specifically focusing on NMs are required to substantiate this.\n\n\nOther exposure routes\n\nSeveral routes of administration have been discussed above to outline how zebrafish may be exposed to NMs to mimic pulmonary, dermal and oral exposure and intravenous injection. Other administration routes can also be used that do not necessarily mimic a specific route of human exposure but can nevertheless be used to screen the inflammogenicity of NMs (and other substances/pathogens). Importantly, the use of a wider range of exposure routes provides an opportunity to make testing more ethical by promoting the use of non-protected life stages of zebrafish.\n\nThe otic vesicle is the ear of the zebrafish and is a closed hollow cavity that is usually devoid of immune cells (Haddon and Lewis 1996, Harvie and Huttenlocher, 2015). An infiltration of leukocytes (e.g., neutrophils, macrophages) into the otic vesicle has been observed following microinjection of different stimuli such as CXCL-8, fMLF, LTB4, LPS, and bacteria (Harvie and Huttenlocher 2015, Benard et al., 2012, de Oliveira et al., 2013). In our work, we have demonstrated that injection of Ag NMs into the otic vesicle of early life stages of zebrafish (3 dpf) stimulated an infiltration of neutrophils to this site (Gillies et al., 2022). However no other studies could be identified that have investigated the toxicity of NMs following their microinjection into the otic vesicle. Benard et al., (2012) have provided guidance on how to inject pathogens into the otic vesicle and monitor inflammatory responses, which can be adapted for NMs. When performing a microinjection into any site in zebrafish embryos/larvae it is recommended that a tracer molecule (such as dextran) is used to confirm the successful injection of the target site. Based on our own studies (Gillies et al., 2022) we suggest that injection into the otic vesicle of transgenic zebrafish offers an effective way to assess for inflammatory responses to NMs.\n\nBrain ventricles are cavities in the brain of zebrafish that contain cerebrospinal fluid (Gutzman and Sive 2009). Inflammatory responses to range of pathogens (e.g., bacteria (Benard et al., 2012, Rocker et al., 2015 Jim et al., 2016, Du et al., 2017, Mazon-Moya et al., 2017), and fungi (Brothers and Wheeler, 2012, Torraca et al., 2015, Voelz et al., 2015)) have been investigated in transgenic zebrafish (with fluorescent immune cells) following their microinjection into the hindbrain ventricle. The hindbrain ventricle is initially devoid of leukocytes, but over time (from 35 hpf) there is a progressive increase in the number of resident immune cells (such as macrophages (microglia)) (Herbomel et al., 2001). This has sometimes led to investigators selecting other exposure sites which are devoid of leukocytes when investigating inflammatory responses to different stimuli (Harvie and Huttenlocher, 2015) as the quantification of the inflammatory response is easier to perform in leukocyte-free regions. However, macrophages are resident in the organs (e.g., lungs) of rodents and humans, and thus it may be more physiologically relevant to conduct studies in zebrafish where there are resident tissue macrophages. In our searches of the literature, no studies were identified that have investigated NM mediated inflammatory responses following their microinjection into the hindbrain ventricle of zebrafish so this exposure site should be considered in future studies and the use of non-protected life stages prioritised to promote alignment with the 3Rs principles. Guidance is available on how to perform microinjections into the hindbrain ventricle for pathogens, which could be modified for application to NMs (Benard et al., 2012, Brothers and Wheeler, 2012).\n\nTransection of the tail fin of non-protected life stages of zebrafish (typically at 3 dpf) causes a wound that stimulates a rapid and robust inflammatory response at the injury site. The inflammatory response to tail fin injury was first characterised by Renshaw et al. (2006), who investigated neutrophil responses to injury over time. Whilst the tail fin injury model has been most commonly used to investigate tissue repair and regeneration in vivo, the model can also be used for a variety of other purposes. For example, the tail fin injury model has been used to investigate the efficacy of anti-inflammatory drugs that promote the resolution of neutrophilic inflammation (e.g., Hoodless et al., 2016, Lucas et al., 2013, Rahman et al., 2019) and to investigate inflammatory responses to chemicals and pro-inflammatory stimuli, such as lipopolysaccharide (LPS) (Cordero-Maldonado et al., 2013).\n\nFor the first time, we investigated whether aqueous exposure of larval transgenic zebrafish (with fluorescent immune cells) to NMs could enhance the inflammatory response that was activated following a tail fin injury (Gillies et al., 2022). We found that the neutrophil response at the injury site in zebrafish was enhanced and took longer to resolve following aqueous exposure to Ag and ZnO NMs relative to the untreated (injured) controls (Gillies et al., 2022). More recently, we have also investigated neutrophil and macrophage responses to ultrafine carbon black (ufCB) using the tail fin injury model. ufCB is a carbon particle (14 nm diameter) that is commonly used as a surrogate to represent particulate air pollution (PM10) in toxicology experiments. PM10 is known to cause a spectrum of adverse health effects and inhalation of ultrafine particles (diameter of <100 nm) in PM10 are likely associated with the activation of inflammation and oxidative stress in the lungs and other sites (reviewed by Johnston et al., 2018). There is evidence from rodent and in vitro studies that ufCB can stimulate a pulmonary inflammatory response (e.g., Li et al., 1996). Thus, we investigated whether the pro-inflammatory effects of ufCB could be assessed in transgenic zebrafish. We observed that the inflammatory response activated in injured zebrafish is enhanced following exposure to ufCB, as indicated by the increased number of neutrophils and macrophages which accumulate in the injury site compared to the untreated injured control (data not shown). This finding and evidence presented in Gillies et al. (2022) therefore demonstrate the utility of the tail fin injury model in investigating NM mediated inflammatory responses.\n\nThe tail fin injury is easy to perform and injured zebrafish can be exposed to NMs (or other substances/pathogens) via water to investigate the dynamics of the inflammatory response that is activated. The tail fin injury zebrafish model therefore offers an approach that allows assessment of whether NMs activate an inflammatory response in a relatively fast, efficient way and is not as technically challenging as performing microinjections, enabling it to be more readily widely adopted.\n\nGuidance is available on how to perform a tail fin injury (e.g., Lisse et al., 2015), however we have observed that there are differences in the magnitude and the duration of the inflammatory response that is activated in the injury site of zebrafish in published studies (Table 1). There are several factors which may influence why different studies show different results including: the severity of the injury, the region used to quantify the inflammatory response, the strain of zebrafish used, the time points investigated and sample size used (Table 1). It is therefore prudent to reflect on what aspects of the experimental design may influence the experimental outcome for this model. In this section we focus on the importance of the severity of the injury and region where the responding immune cells are assessed, with other factors considered later.\n\nStudies are presented in alphabetical order. The number of larvae included in each group is provided, as well as information on how many independent experiments were included, when available. The mean neutrophil count is provided for each time point, and the neutrophil counts are presented from the shortest to longest time point investigated. The size of the region that was used to perform the neutrophil counts is provided when stipulated in the publication. The numbers of neutrophils accumulating in the injury site are not typically stated in the text of research papers and so the number of neutrophils was often extrapolated from the graphs presented and therefore may not be fully accurate.\n\nThe severity of the injury that is inflicted to the tail fin can markedly influence the inflammatory response that is activated (Elks et al., 2011). We have observed that studies do not consistently report on the severity of the injury used, which is an important omission from the methodology. The blood vessels that make up the circulatory loop (where the caudal artery loops back into the caudal vein) can be used as a marker for amputating the tail fin to induce a severe injury, whereby the notochord is transected (Figure 3), as established by Renshaw et al. (2006). A less severe, more moderate injury can be induced whereby the tip of the notochord is used as the marker for transection, avoiding damage to the notochord and blood vessels (Figure 3) (Barros-Becker et al., 2017, Hoodless et al., 2016, Li et al., 2012, Lucas et al., 2013). A minor injury can be inflicted through making a small tissue nick in the tail fin (Elks et al., 2011; Paredes et al., 2019; Tauzin et al., 2014). We suggest that a moderate injury is employed when investigating NM toxicity in injured zebrafish to ensure the inflammatory response initiated is robust (i.e., can be detected) but avoids damage to critical structures such as the blood vessels or the notochord, as the latter may make quantification of the response more variable and challenging. Interestingly, different approaches have been used to cause the injury (e.g., the use of a scalpel, needle, or laser), with the use of a scalpel the most common approach (Table 1). There is evidence that burn wounds (thermal injury) can induce a greater inflammatory response than transection using a scalpel (Miskolci et al., 2019).\n\nThe image shows the tail fin of a 3 dpf Tg (mpx: EFGP)114 larvae (with fluorescent (green) neutrophils) prior to tail fin transection. The edge of the circulatory loop (red) (A), where the caudal artery becomes the caudal vein, can be used as a marker to perform a severe tail fin injury (B). The tip of the notochord (yellow) (C) can be used as a marker to perform a moderate tail fin injury (D). A small nick that is removed from the tail fin can induce a minor injury. The blue line is used to indicate the outline of the zebrafish larvae. Image obtained on a Leica M205 FCA fluorescent stereomicroscope using a Leica DFC7000T digital colour camera, at 160x magnification. Scale bar 250 μm. Image taken by Dr Suzanne Gillies.\n\nWhilst some published studies have reported what region at the injury site was used when counting immune cell accumulation, many do not (Table 1). Differences in the counting region used in different studies may also help explain why there are differences in the numbers of neutrophils which accumulate at the injury site in the published literature (Table 1). Accordingly, it is recommended that a standard region for counting the immune cell response region is identified, and in the meantime published studies ensure this information is reported.\n\n\nSummary of exposure routes\n\nWe have outlined what routes of administration can be used to expose zebrafish to NMs to provide potential mammalian surrogates for exposure via the lungs, skin and intestine and intravenous injection. We have also discussed what other exposure routes might be considered when assessing whether NMs activate an acute inflammatory response but which are not relevant to a particular exposure route in humans/rodents. A summary of all exposure routes discussed is presented in Figure 4.\n\nAqueous exposure of zebrafish to NMs can lead to exposure via the gills, skin or intestine (via drinking or the diet) to investigate responses following respiratory, dermal and oral exposure respectively. Exposure of injured zebrafish to NMs via water can allow for acute inflammatory responses at the injury site (as well as other target sites) to be monitored to screen their toxicity. Microinjection of NMs into the otic vesicle, swim bladder, hindbrain ventricle, caudal vein or yolk sac can allow local and systemic inflammatory responses at the injection site and/or whole body to be investigated. Created using BioRender.com.\n\nThe choice of exposure route for nanotoxicology studies that use zebrafish to investigate the impacts of NMs on human health is likely to be dictated by several considerations, including, but not limited to, the following:\n\n1. The anatomical relevance of the exposure site in zebrafish to the human/rodent exposure route or target site of interest.\n\nIt is key to consider whether the exposure route selected in zebrafish is relevant to how humans/rodents would be exposed. However, whilst some exposure routes for zebrafish are not directly relevant to a specific route of human/rodent exposure they can still be considered as they can be used to screen NM toxicity and may have ethical benefits (e.g. as they allow for the use of non-protected life stages of zebrafish).\n\n2. The requirement to assess responses following local or systemic administration, and the need to investigate local and/or systemic responses.\n\nExposure via microinjection allows for targeted administration of substances to specific sites. Alternatively, exposure via water or intravenous injection is often associated with a more widespread distribution of the test substance. The toxicity of NMs may be restricted to the exposure site, or effects may be observed at sites distal to the point of exposure. Rodent studies often focus on investigation of local inflammatory responses that are activated at the exposure site but may also consider whether systemic responses are also stimulated. For example, responses in extra-pulmonary sites such the liver may be investigated following the pulmonary exposure of rodents. Importantly, the site of exposure used for a pathogen/substance in zebrafish will dictate whether the infection/exposure remains localised or can become systemic (Benard et al., 2012). Thus, the requirement to assess local and/or systemic responses should be considered when selecting an exposure route for zebrafish.\n\n3. The life stage of zebrafish that will be used.\n\nEarly life stages of zebrafish (<5 dpf) are not protected, and thus it is often desirable to work with early life stages of zebrafish to make testing more ethical. Administration via the otic vesicle, caudal vein/Duct of Cuvier, skin, yolk sac and hindbrain ventricle and exposure of injured fish via water would typically use non-protected life stages of zebrafish, whereas exposure via the gills, swim bladder and ingestion (water or diet) would employ protected life stages (Figure 4). If protected life stages of zebrafish are used this would require permission from the responsible authorities (e.g., animal licencing and local ethical approval) in the country that the work is performed and their use would not constitute a replacement of animal use. The use of non-protected life stages of zebrafish will restrict what exposure and target sites can be investigated, as systems develop at different rates and times. Whilst there are clear 3Rs benefits of working with non-protected life stages of zebrafish there sometimes also 3Rs benefits of working with protected life stages of zebrafish over rodents. For example, it may be possible to monitor an inflammatory response in the same zebrafish over time in protected life stages leading to reductions in animal use (although this will be dependent on whether the pigmentation of zebrafish obscures visualisation of the target site and whether capser strains are available that lack skin pigmentation).\n\n4. The need to assess responses following single or repeated exposures.\n\nOECD protocols are available to assess toxicity of substances (e.g., NMs) following different exposure routes (such as inhalation, ingestion) in rodents and often require repeated exposure to the substance under investigation. The capacity to perform repeated exposures likely depends on the zebrafish life stage as well as the exposure route that has been selected. For example, whilst repeated exposure to NMs via water is possible, repeated exposures via microinjection are not normally performed and is unlikely to be possible for all microinjection sites, particularly in non-protected life stages.\n\n5. The requirement to access specialised equipment.\n\nMicroinjection is technically challenging, time consuming and requires specialized equipment which may not always be available to researchers, and as a consequence exposure of zebrafish larvae to NMs via water is likely to be the preferred route of exposure. However, it has to be accepted that whilst each individual fish may have the same exposure level to the test substance when exposed via water that the uptake of NMs by individual fish may vary, which is a complicating factor for this exposure route. Accordingly, assessing NM fate in zebrafish following exposure via water (as well as other routes) in parallel to evaluating inflammatory responses is important to interpret the data obtained. The approach used to visualize and quantify the fate of NMs in the zebrafish will be reliant on what NMs are being investigated. For example, to image the fate of fluorescent NMs fluorescent microscopy can be used (e.g., confocal or light sheet microscopy) and for unlabelled NMs other imaging modalities will have to employed (e.g., transmission electron microscopy, CARS microscopy).\n\n\nRecommendations for new users\n\nWe have summarized the findings and experimental design employed in existing studies which have assessed neutrophil responses in zebrafish with a tail fin injury (Table 1) and following the microinjection of different stimuli into the otic vesicle (Table 2) in order to identify similarities and differences in the experimental design used. The tail fin injury model was selected as this has been widely used by different research groups in the published literature to assess inflammatory responses in transgenic zebrafish. Administration of substances via microinjection into the otic vesicle is the most popular route of exposure used in existing studies investigating inflammatory responses to different stimuli (and most commonly pathogens) following microinjection.\n\nStudies are presented in alphabetical order. The number of larvae included in each group is provided, as well as information on how many independent experiments were included, when available. The range of neutrophils counted in the otic vesicle is presented as well as the average count. When available data for the control group is included. The numbers of neutrophils accumulating following microinjection are not always explicitly stated in the text of research papers and so the number of neutrophils was extrapolated from the graphs presented and therefore may not be fully accurate.\n\nWe observed a lot of variation in the design of existing studies, and we will focus on discussing how the following parameters may influence the experimental outcome: zebrafish strain and life stage selected, selection criteria for zebrafish embryos/larvae used in experiments, approach used to quantify the inflammatory response, sample size used, inclusion of positive and negative controls and dose selection.\n\nVarious transgenic strains of zebrafish larvae can be used to assess inflammatory responses to NMs, with neutrophil accumulation most commonly investigated, and fewer studies also investigating macrophage responses. Several transgenic strains have been generated with fluorescently labelled neutrophils, however the Tg (mpx:eGFPi114) strain has been the most popular strain of zebrafish used, to date (Tables 1 and 2). Different strains may vary in their specificity (for neutrophils) and sensitivity, which will likely influence the magnitude of the inflammatory response that is activated. Whilst the Tg (lyz: EGFP) strain has been used to quantify neutrophil responses there is evidence that lyz is also expressed in macrophages and thus these strains may therefore not be suitable when assessing neutrophil responses (Brannon et al., 2009, Hall et al., 2007, Keightley et al., 2014). Transgenic strains which exploit the myeloperoxidase (mpx) promoter (e.g. Tg mpx: EGFP) are therefore more commonly used as the mpx gene is neutrophil-specific (Renshaw et al., 2006, Ruzicka et al., 2019). To investigate macrophage responses, the Tg mpeg:mCherry strain can be used (Ellett et al., 2011). Investigating both neutrophil and macrophage responses in tandem is advantageous and can be achieved using the Tg mpx: EGFP/mpeg:mCherry strain (Davis et al., 2016, Jim et al., 2016, Kaveh et al., 2020). There are also several methods than can be employed to deplete functional macrophages and neutrophils from zebrafish (reviewed by Rosowski, 2020) that may be useful when exploring the role of specific immune cell types in NM mediated inflammatory responses.\n\nWe have focused on the availability of transgenic strains that allow neutrophil and macrophage responses in zebrafish to be investigated. Other researchers may be interested in other immune cell types. For example, transgenic strains (e.g. gata2:eGFP) have been developed to investigate the role of eosinophils in inflammatory responses (e.g. Balla et al., 2010). The adaptive immune system does not develop until ~four weeks in zebrafish and thus the requirement to assess adaptive immune responses should inform what life stage of zebrafish is used. At this life stage the fish skin will no longer be transparent and thus the approaches discussed earlier to remove skin pigmentation will have to be used. Alternatively strains of zebrafish (with fluorescent immune cells) are available which are genetically modified to lack pigmentation and allow inflammatory responses to be monitored for a longer duration. Histology may also be performed to assess inflammatory responses in later life stages.\n\nThe strains already discussed are focused on investigating the accumulation of immune cells in specific sites following NM exposure, however other aspects of the inflammatory response may be of interest to explore using other transgenic strains. For example, the reporter strain Tg (NFκB:EGFP) could be utilised to assess the activation of the pro-inflammatory transcription factor NFκB (Kanther et al., 2011). In addition, the activation of oxidative stress by NMs can be investigated via the visualization of the electrophile-response element (EpRE), a promoter region located on nuclear factor erythroid 2–related factor 2 genes (Nrf2), using the Tg (3EpRE:hsp70:mCherry) strain (Mourabit et al., 2019). Furthermore, a strain with a hydrogen peroxide (H2O2) sensor has been developed and has been used to investigate the role of this reactive oxygen species (ROS) in leukocyte recruitment (Niethammer et al., 2009).\n\nStudies do not consistently report what criteria are used for zebrafish embryo/larvae selection. It is important to select healthy zebrafish embryos/larvae to ensure they are free from physiological abnormalities and that they are at the expected stage of development. Given the rapid rate of development of larval zebrafish, it is recommended that researchers ensure that life stages have been screened according to Kimmel et al. (1995) so that experimental cohorts are at the same developmental stages. If using transgenic strains it also imperative to ensure that they have a robust population of the fluorescently labelled cells of interest (e.g., macrophages and/or neutrophils). It is therefore important to select a consistent population of larvae to ensure obtained data are robust and comparable. Screening larvae to ensure that consistent numbers of the cells of interest are present can be done by visually assessing the larvae (using fluorescent microscopy) prior to the start of the experiment. However, this approach is subjective. Fluorescence based approaches, such as measuring the mean FI of the larvae using imaging software (e.g., Iplab or ImageJ) could be used to ensure the selected larval population fall within a user-defined range. Screening methods are not often well defined or reported currently in the published literature. It is therefore our recommendation that this information is provided in publications to ensure that data can be accurately compared between studies.\n\nThe sample size is the number of experimental units that have been included for each (control and treatment) group (Lazic et al., 2018). However, the experimental unit is not always clearly reported in the published literature which use zebrafish embryos/larvae (Tables 1 and 2). This is an important omission as if the experimental unit is not defined appropriately then the sample size can be over-estimated which can impact on the statistical analysis that is performed and interpretation of the data (Lazic, 2022). As an example, for aqueous exposure, zebrafish embryos/larvae are typically exposed in microplates (e.g., 96, 24 or 12 well plates), but the number of larvae contained in each well is not often stipulated in published papers. If one zebrafish embryo/larva was added to each well and different treatments were added to different wells of the same plate, without cross-contamination between wells, then each zebrafish embryo/larva can be considered an experimental unit, and each embryo/larva contributes one data point to the analysis. However, if more than one zebrafish embryo/larva was added to each well then each well would represent an experimental unit as embryo/larva from the same well are not independent and cannot be allocated to different treatments. In this case, to avoid pseudoreplication, measurements taken from an individual embryo/larva can be averaged for each well so that each well provides one data point to the analysis (Lazic, 2010). Researchers typically use <40 embryos/larvae for each (control or treatment) group (Tables 1 and 2), and it is therefore assumed that independent experiments are performed, but it is unclear whether this is always the case and how many embryo/larvae are included in each experiment. In this case, using a block design would ensure that all treatment and control groups are included each time the experiment is performed, which would increase the chances of detecting an effect (Festing, 2014). Furthermore, it is not common for researchers to indicate whether the position of control and treatment groups are randomized on a microplate. This is particularly important if there is potential for contamination of the test substance between wells. Therefore, it is recommended that researchers provide more detailed information on their experimental design when describing their methodology to ensure that the issues discussed above are addressed.\n\nPower calculations can be performed to identify what sample size should be used but these are currently based on a limited data set. Therefore, when the model has been used more widely it is likely that more accurate estimations of the variability to be expected with this model will be available to calculate the sample size.\n\nThe number of zebrafish embryo/larvae that can be used in each experiment is limited by several factors such as; the number of fertilized embryos produced during spawning, the pre-screening process used to select zebrafish embryos/larvae for inclusion in the study (as this dictates how many embryos/larvae are available to the researcher), the time taken to perform the experiment (e.g., anaesthetizing larvae, inducing a tail fin injury or microinjecting larvae, acquiring images, and larval recovery time), the number of treatment and control groups desired, and the number of researchers performing the experiment (as this will impact on equipment availability). These limiting steps should be taken into consideration when designing experimental work to ensure it is feasible. For example, imaging time points should be set at practical intervals to reflect the time needed to acquire images of all the larvae in all treatment and control groups.\n\nIn zebrafish larvae, macrophages are the first immune cells to appear and which occur from 22 hpf (Rosowski, 2020) and neutrophils are apparent from 2 dpf (Lieschke et al., 2001). Therefore, when assessing inflammatory responses in larval zebrafish, experiments can start from 2 dpf. However, at this stage the chorion is still present and must be removed either manually or chemically to ensure exposure of the zebrafish to the NMs. Dechorionating embryos is time consuming and can damage the larvae (i.e., the process itself can cause inflammatory response that may interfere with the experiment). Thus, most studies performed to date (Tables 1 and 2) have exposed zebrafish after they have hatched.\n\nFor the tail fin injury model and for microinjection studies using the otic vesicle the age of zebrafish used is typically 3 dpf (Tables 1 and 2). The zebrafish larvae are non-protected until 5 dpf, thus starting at 3 dpf allows for inflammatory responses to be monitored for up to 48 hours in non-protected life stages. Using zebrafish at this life stage also means that zebrafish will have hatched from the chorion naturally prior to beginning experiments. The use of later time points would require the use of protected life stages of zebrafish which would have more ethical implications and require permission from responsible authorities. However, the duration of the experiment will be limited by the transparency of the fish, as it becomes more challenging to monitor the inflammatory response as skin pigmentation increases (see above).\n\nTo assess whether an inflammatory response is activated in zebrafish, the accumulation of immune cells to the target site of interest needs to be visualized and quantified. Histological examinations have been used to visualize inflammatory responses to NMs in wild type zebrafish strains, however, here we focus on how to quantify responses in transgenic zebrafish strains with fluorescently labelled immune cells. In order to quantify inflammatory responses, images are typically taken in the same larvae at different time points using fluorescent microscopy (e.g., stereomicroscopy, confocal microscopy, light sheet microscopy) and immune cells are counted in the region of interest (e.g. injury or injection site and/or whole organism). Depending on the target site of interest, imaging modalities that enable long-term imaging without photo-bleaching and permit 3D image reconstruction may be employed such as selective plane illumination microscopy (SPIM) (Taylor et al., 2019). Several published studies have investigated responses at only one time point (Tables 1 and 2). However, to capture the initiation, peak, and resolution (or lack thereof) of inflammation in non-protected life stages of zebrafish, it is recommended that multiple time points are used as this allows the dynamics of the inflammation response to be investigated. When imaging at set time points larvae are most commonly placed on a glass microscope slide (in the presence or absence of agarose) to capture the required image. The use of microfluidic devices may also be useful when imaging discrete regions such as the otic vesicle, as these enable the immobilisation of the larvae in the desired position (e.g., Bischel et al. 2013, Ellett and Irimia, 2017a, 2017b). It is recommended that images of the whole larvae as well as the region of interest (e.g., otic vesicle, swim bladder, gill, injury site) are taken as the NMs may elicit a local and systemic inflammatory response (Gillies et al., 2022). In addition, if possible, it would be useful to take images at different focal planes (z stacks) to ensure the images capture cells present in three dimensional regions of interest such as the otic vesicle or swim bladder, which would require access to confocal or light sheet microscopy (e.g., Logan et al., 2018, Winter et al., 2017).\n\nThe accumulation of cells can be quantified by performing manual or automated counts, often using software such as Fiji (ImageJ) or Imaris. Manually counting cells is time consuming and more subjective, however, it is a commonly used technique when quantifying cellular responses in zebrafish larvae (Loynes et al., 2010, Miskolci et al., 2019, Gratacap et al., 2014). The error involved in manual counting can be reduced by having at least two people to quantify the responses to confirm findings, and via experimental blinding, such as the method described by d’Alençon et al., (2010). Automated software can be exploited to facilitate rapid and accurate cell counts, such as cell counting macros/plugins that can be used in software such as ImageJ (e.g. Renshaw et al., 2006, Renshaw and Ingham, 2010, Wittmann et al., 2012). Automated counting requires validation (e.g., the user should perform some manual counts to confirm the data), however existing studies have shown good correlation between automated and manual counts (Renshaw et al., 2006). Alternatively, the inflammatory response can be quantified via fluorescence intensity-based approaches. For example, by measuring the fluorescence intensity (FI) of the region of interest rather than counting individual cells, an increase or decrease in FI can be used to indicate an increase or decrease in the numbers of immune cells (e.g. Duan et al., 2016).\n\nWhilst imaging at set time points is useful for the quantification of the inflammatory response, it may be advantageous to acquire time-lapse video imaging which has the added benefit that it can enable the tracking of immune cells (Feng et al., 2010, Kaveh et al., 2020). To maintain the zebrafish larvae in a specific position for the duration of time-lapse imaging, they can be mounted on glass microscope slides using low melting point (LMP) agarose (e.g. Hirsinger and Steventon, 2017). This technique is not appropriate for long periods of time, as it requires the use of a heated microscope stage to allow for normal embryo development, and the agarose may have to be replaced/adjusted throughout the experiment to accommodate growing larvae. Guidance is available in the published literature on how to overcome this issue (e.g. Hirsinger and Steventon 2017).\n\nZebrafish embryos/larvae have been used widely to assess NM (eco) toxicity, as such toxicity data (i.e., mortality, growth, development) for many NMs have been published, which can inform the selection of non-lethal doses for immune effect directed studies. For example, LC50 values (the concentration required to kill 50% of the exposed population) are known for NMs from the JRC repository (which are widely studied by the international scientific community) including for example Ag (e.g. NM300K) and ZnO (e.g. NM110) (Massarsky et al., 2013, Muth-Köhne et al., 2013, Osborne et al., 2013, Küçükoğlu et al., 2013, Choi et al., 2016). When using transgenic zebrafish larvae to assess inflammatory responses to NMs of known toxicity, appropriate sub-lethal concentration ranges can therefore be determined from the existing data derived from aquatic toxicology studies using wild type zebrafish embryos/larvae. If data are not available for the NM of interest then it is advised that dose finding studies should be performed as part of pilot work.\n\nWhen working with zebrafish larvae it is difficult to maintain a sterile environment. It is therefore possible that the inflammatory responses in larvae may be influenced by contaminants (e.g., endotoxin (LPS)) present in the exposure medium. However, existing studies often neglect to report what controls are included. Brown et al. (2007), found that in the absence of an infectious agent, a tail-nick wound would not elicit a moderate inflammatory response. This study highlights a need to include appropriate negative controls and to better monitor the presence of microbes and their products within the system water from which the larvae are cultured as such factors may affect leukocyte recruitment. The microbial environment must therefore be acknowledged as a potential confounding variable which has a potential impact on aspects of the immune response such as recruitment and cytokine expression.\n\nThe inclusion of positive controls demonstrates that you are able to detect the response of interest (e.g., inflammation) within your test model. We assessed inflammatory responses to several positive controls in both the tail fin injury and otic vesicle microinjection experiments (Gillies et al., 2022) as we observed that existing studies neglected to consistently include a positive control. More specifically, we evaluated what inflammatory response were activated by the pro-inflammatory compound LPS and the chemoattractants fMLF, LTB4, C5a, and CXCL8. Our findings suggested that the positive control required will depend upon the route of exposure (Gillies et al., 2022). For example, LTB4 was identified as the most appropriate positive control for tail fin injury model and CXCL8 was the most effective substance at stimulating an inflammatory response following microinjection into the otic vesicle.\n\nFor aqueous exposure of injured fish, NMs are typically suspended in zebrafish water of various types (e.g., E3, OECD medium). It is therefore essential to include zebrafish medium as a negative control to quantify the inflammatory response in the absence of NMs (or the test substance of interest). As the microinjection itself may cause an injury that stimulates an inflammatory response, it is crucial to include an appropriate negative and vehicle control (e.g., PBS in the absence of the test substance). For comparative purposes it is also useful to include a group with has not been injected.\n\n\n3Rs benefits\n\nA PubMed search (Oct 2022, search terms: nanomaterial OR nanoparticle AND lung AND rat or mouse AND inflammation AND neutrophil) revealed that >300 rodent studies have assessed pulmonary inflammatory responses to NMs, with a higher proportion of existing studies using mice as the test model. Such studies typically assess the toxicity of a panel of NMs (average of three, ranging from one to 13), administer more than one dose (average of two, ranging from one to seven) and assess inflammation at several time points (average of three, ranging from one to six). The average group size used for mouse studies is eight (with an average of 75 animals used/study), and for rats the average group size is six (with an average of 65 animals used/study). Existing studies have typically used only one sex of rodents, although some studies have used both male and female animals (e.g., Wan et al., 2017, Srinivas et al., 2012). In addition, one route of administration is typically used, with intratracheal instillation being prioritized, although some studies have compared the response observed following two routes of exposure (e.g. intratracheal instillation and inhalation (Morimoto et al., 2016, Silva et al., 2014)). The variety of NMs currently in use or development in addition to the diversity of future generations of NMs means there are a large number (tens of thousands) of NMs whose safety will need to be assessed. Therefore, it is not sustainable to rely on rodent testing for nanotoxicology testing and we suggest that the increased use of zebrafish could offer many 3Rs benefits. For example:\n\ni) Non protected life stages of zebrafish could be used to screen NM toxicity as an alternative to using rodents leading to a replacement of animal (rodent) use. For example, tests performed using early life stages of zebrafish could identify highly toxic NMs at an early stage of innovation whose toxicity would not be tested further in rodents.\n\nii) Non protected life stages of zebrafish could be used as a bridge between in vitro and rodent studies to help inform the design of rodent studies and more specifically to:\n\na. Help prioritize NM selection for more extensive testing in rodent models, thereby leading to a reduction in rodent use. This is particularly important as nanotoxicology rodent studies frequently test the toxicity of a panel of NMs and data obtained from zebrafish could be used to prioritise NM selection for in vivo (rodent) testing.\n\nb. Allow testing of fewer doses of each NM in rodents, leading to a reduction in rodent use as data from a whole organism (zebrafish) has already been obtained to investigate NM toxicity.\n\nc. Help refine rodent studies as high toxicity NMs could be identified and this knowledge can inform dose selection for rodent studies to avoid administration of highly toxic doses to rodents.\n\niii) Inflammatory responses can be monitored in the same organism over time leading to a reduction in animal use as separate treatment and control groups are not required for each time point of interest.\n\nIn the short term we anticipate that the use of zebrafish will precede rodent work to identify whether rodent testing is needed and to help inform the experimental design of rodent studies. This would require that a tiered testing strategy is employed to assess NM toxicity. Such testing strategies would progress from applying simple and advanced in vitro models, before employing (non-protected life stages of) zebrafish and finally rodent testing would only be performed if deemed essential e.g., for regulatory approval (e.g., Johnston et al., 2018, Stone et al., 2020). The use of zebrafish in nanotoxicology is still in its infancy. Once the model has been adopted more widely, and standard protocols have been developed that allow testing to be performed in a harmonized manner, it is possible that zebrafish may replace rodent testing when investigating NM mediated inflammatory responses (as an indicator of their toxicity) in the longer term.\n\nWe have focused on how zebrafish can be applied to assess inflammatory responses to NMs to screen their toxicity but inflammatory responses to other substances could be investigated in this model and other transgenic strains could be used to assess other adverse responses to NMs.\n\n\nConclusions\n\nInvestigation of the capacity of NMs to stimulate an inflammatory response is commonly used to screen their toxicity in in vitro and in vivo (rodent) models. Transgenic zebrafish offer an effective alternative (i.e., non-mammalian) model to assess whether NMs activate inflammatory responses when assessing their safety. Indeed, we have successfully used non-protected life stages of transgenic zebrafish with fluorescent neutrophils (e.g., Tg (mpx: EGFP114) to investigate inflammatory responses to NMs and thereby demonstrated their usefulness as a model for evaluating NM toxicity. More studies are now required to test a wider panel of NMs, to explore more administration routes and to assess the utility of other transgenic strains.\n\nWe have outlined how zebrafish may be exposed via water or microinjection to NMs to reflect exposure routes that are relevant to human/rodent exposure. As not all systems that are relevant to human and rodent exposure are fully developed in non-protected life stages of zebrafish the use of a wider range of exposure routes has been explored as their use provides an opportunity to make testing more ethical. More specifically, we have highlighted exposure routes that can be employed in non-injured or injured non protected life stages of zebrafish to screen NM toxicity. The experimental design employed in existing studies to investigate inflammatory responses to a range of pathogens and stimuli in zebrafish is diverse and can have a profound impact on the experimental outcome. It is therefore essential for researchers to justify their approach and to provide sufficient detail on the experimental design employed so that data can be interpreted correctly and to allow comparisons to be made between studies. Ideally, standard methods will be developed that allow the inflammatory response activated by NMs (and other chemicals, pharmaceuticals and pathogens) to be assessed in a consistent manner The increased use of zebrafish in nanotoxicology is likely to enhance implementation of the 3Rs principles to make testing more ethical, cheaper, quicker and potentially more predictive. Whilst a focus has been placed on the use and application of transgenic zebrafish for assessment of pro-inflammatory responses activated by NMs in this paper the themes discussed are not restricted to NMs but relevant also to their appliction in ecotoxicology or human health focused studies investigating other substances (e.g. chemicals, pharmaceuticals) and pathogens as well as for studies investigating disease pathogenesis.",
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}
|
[
{
"id": "326092",
"date": "11 Oct 2024",
"name": "Stephen Renshaw",
"expertise": [
"Reviewer Expertise Zebrafish models of innate immunity"
],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 summarises different approaches of exposure within zebrafish to investigate the inflammatory response to nanomaterials (NM). It is specifically aimed at researchers new to using zebrafish. The authors provide a good introduction to using zebrafish as a model organism and how it can contribute to the 3Rs. They give essential detail on key aspects to think about when using the zebrafish as a model and screening tool that will promote experimental design discussions amongst new users. The review has 2 main sections: it addresses NM delivery and then more specifically, how to study the inflammatory response. Some transgenic lines are mentioned that are key to investigating the inflammatory response in vivo but there are several missing (see sections below). The language used is clear and accessible and the review uses nice diagrams to summarise information. Occasionally, some paragraphs are very brief and feel oddly placed, without a connection or explanation as to why the statements are there. The information given is accurate and mainly cited correctly and thoroughly. Other reference suggestions are listed below.\nReferences Some key references are missing as detailed below: Section: Zebrafish and Inflammation\nPage 4, 1st paragraph – 2 groups first identified granulocytes in zebrafish around the same time and both should be cited. Lieschke GJ, et al., 2001 (Ref 1)\nBennett CM, et al., 2001 (Ref 2)\nPage 5 – Include fms: GFP line (Dee, 2016) and mfap4 lines (Walton 2016) as alternatives to mpeg lines to study macrophages. Both are useful depending on what aspect of macrophages the researcher is investigating.\nDee CT, et al., 2016 (Ref 3)\nPage 22. Reference for automated analysis is from the Grabher group not Renshaw and Ingham 2010, which is a secondary source.\nd'Alençon CA, et al., 2010 (Ref 4)\nNomenclature Our own early manuscripts did not use correct nomenclature and we are grateful to the reviewers who pointed us in the right direction. The nomenclature of the transgenic lines is not correct in this review (according to zfin nomenclature conventions) i.e. all italics, state if a BAC was used, allele numbers should not be superscript. Allele numbers are not included for many of the lines suggested and as this review is directed at new users it would be very informative to include these. E.g. TgBAC(mpx:GFP)i114 In the legend for Figure 1, this compound transgenic should be TgBAC(mpx:GFP)i114; Tg(mpeg:mCherry) -adding the allele code for the mpeg line in italics. This approach should be used for all transgenics, as set out by ZFIN nomenclature conventions.\nSection details: Research highlights. “Zebrafish could provide better predictions of the impact of substances on human health than rodents” No evidence is provided for this model being better than rodents. It seems likely there are some ways that this model might have advantages (for example 3Rs) but others (tissue specificity etc) where rodents might be more beneficial. I would suggest a more nuanced discussion.\nIntroduction Page 4. “In the EU, early life stages of zebrafish, prior to exogenous (independent) feeding at 120 hours/5 days post fertilisation (hpf or dpf)), are not protected by law and as such do not require permission from responsible authorities to be used in scientific research (EU Directive 2010/63/EU).” I’m not clear this is completely correct. Although the larvae are not “protected” under UK law, their transgenic parents are, meaning that the experiments are protected by law. I would also consider the generation of a genetically modified larvae to be a regulated procedure. I would suggest this paragraph is revised after appropriate discussion.\nNanomaterials and inflammation Page 6, second sentence would be useful to have a reference for this where it is stated that the properties exhibited at nanoscale differ drastically to larger forms.\nZebrafish and nanotoxicity Page 7, 2nd paragraph. I think more needs to be explained as to why transgenic fish are advantageous for this specific study of NMs, over the WT fish stated in the first paragraph, especially in terms of studying the inflammatory response. If the Ecotoxicity and Inflammation sections were combined on page 7 it would resolve this issue and flow better. The first 2 paragraphs seem disjointed so a rewrite of the 3 paragraphs together would make this section clearer.\nAqueous exposure Page 9, first paragraph – talks about limitations of increased pigment development. Could you talk about nacre or casper at this point as an option? Zebrafish gills are quite unlike human lungs in many ways. For balance a discussion of the differences to human lungs would be helpful here.\nTailfin injury model Page 12, 5th paragraph, reference Elks et al 2011 doesn’t show that the severity of injury inflicted influences the inflammatory response that is activated. The same reference is also used to cite the method of a small nick in the tail fin. Again, this isn’t in this ref. This paper isn’t cited in Table 1 either.\nLayout/order The section starting on page 8 to page 11 (Selection of Exposure Route) doesn’t flow. The section of Other Exposure Routes really could fit under the other main headings. Pulmonary exposure\n\nAqueous\n\nMicroinjection – swim bladder…add otic vesicle and hindbrain here. Oral exposure Dermal exposure Intravenous exposure Inclusion of ‘Tail fin injury model’ in this subsection of ‘other routes of exposure’ doesn’t fit. This should be its own section about how to monitor the inflammatory response following one or more of these exposure routes. Yolk sac injection is included within the section on intravenous exposure, which is incorrect.\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": "327733",
"date": "11 Oct 2024",
"name": "Sijie Lin",
"expertise": [
"Reviewer Expertise Zebrafish toxicology",
"nanotoxicology"
],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis is a timely and comprehensive report on using transgenic zebrafish for immune response to nanomaterials. Just a couple suggestions for the authors to consider. 1. The use of zebrafish to \"enhance the implementation of the 3Rs principles\" may still be up for debate? As zebrafish, especially after 4-5 days, is considered as an intact animal. 2. On Scientific benefit: \"Zebrafish could provide better predictions of the impact of substances on human health than rodents\", \"better predictions\" might be a bit too exaggerated, faster or quicker would be better. 3. Reports using transgenic zebrafish to understand the immune responses of engineered nanomaterials are recommended for the authors to consider [Ref-1], [Ref-2].\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/12-51
|
https://f1000research.com/articles/10-409/v1
|
21 May 21
|
{
"type": "Software Tool Article",
"title": "Sherlock: an open-source data platform to store, analyze and integrate Big Data for biology",
"authors": [
"Balazs Bohar",
"David Fazekas",
"Matthew Madgwick",
"Luca Csabai",
"Marton Olbei",
"Tamás Korcsmáros",
"Mate Szalay-Beko",
"Balazs Bohar",
"David Fazekas",
"Matthew Madgwick",
"Luca Csabai",
"Marton Olbei",
"Mate Szalay-Beko"
],
"abstract": "In the era of Big Data, data collection underpins biological research more so than ever before. In many cases this can be as time-consuming as the analysis itself, requiring downloading multiple different public databases, with different data structures, and in general, spending days before answering any biological questions. To solve this problem, we introduce an open-source, cloud-based big data platform, called Sherlock (https://earlham-sherlock.github.io/). Sherlock provides a gap-filling way for biologists to store, convert, query, share and generate biology data, while ultimately streamlining bioinformatics data management. The Sherlock platform provides a simple interface to leverage big data technologies, such as Docker and PrestoDB. Sherlock is designed to analyse, process, query and extract the information from extremely complex and large data sets. Furthermore, Sherlock is capable of handling different structured data (interaction, localization, or genomic sequence) from several sources and converting them to a common optimized storage format, for example to the Optimized Row Columnar (ORC). This format facilitates Sherlock’s ability to quickly and easily execute distributed analytical queries on extremely large data files as well as share datasets between teams. The Sherlock platform is freely available on Github, and contains specific loader scripts for structured data sources of genomics, interaction and expression databases. With these loader scripts, users are able to easily and quickly create and work with the specific file formats, such as JavaScript Object Notation (JSON) or ORC. For computational biology and large-scale bioinformatics projects, Sherlock provides an open-source platform empowering data management, data analytics, data integration and collaboration through modern big data technologies.",
"keywords": [
"Software",
"Computational biology",
"Network biology",
"Systems biology",
"Data lake",
"big data",
"data management",
"data integration"
],
"content": "Introduction\n\nMost bioinformatics projects start with gathering a lot of data. Often bioinformaticians have to work on bespoke datasets, for example, gene expression or mutation data, but in almost all cases this requires some sort of external reference data. The vast majority of projects may call for genome annotations, gene ontologies, tissue-specific expression datasets, drug-related databases, and many other, particular reference datasets. One of the reasons why we use the term’bioinformatics’ in the first place is because we cannot correlate and process all these datasets manually, we need the help and the power of the computers and databases (Greene et al. 2014). Thanks to the current technical advancement, many solutions exist worldwide to take advantage of the available computational possibilities (Marx 2013).\n\nCloud storage solutions such as Amazon’s S3 (https://aws.amazon.com/s3/) or Google Cloud Storage (https://cloud.google.com/storage) offer scalability and flexibility to the matching compute solutions. More importantly, they allow large and potentially shared datasets to be stored on the same infrastructure where large-scale analyses are run. Some companies have utilized cloud resources to offer storage, access to shared datasets, and transparent sharing of data. Cloud storage also provides enhanced reliability, as the data is backed up in several geographical locations (Kasson 2012). However, in most of the cases, these cloud storage companies only offer data storage solutions, but this does not include platforms to execute or work with data.\n\nTo address these issues, we repurposed concepts and open source tools from the top players of the software industry, like Facebook, Netflix and Amazon. With the aim to replace the tedious process of manual data collection before the first steps of any bioinformatics project, we developed a new platform, Sherlock. The Sherlock platform has two main parts: 1) a query engine, which is responsible for executing the given Structured Query Language (SQL) queries, which is the most extensively used database language; 2) a Data Lake, required for data storage, which consists of multiple different databases or datasets, where the Query engine can executes queries against the data. The combination of these two parts streamlines efficient data collection, data integration, and data preprocessing for a given project (Khine & Wang 2018).\n\n\nImplementation and Operation\n\nSherlock is an open source and freely accessible platform that combines several different software technologies together (https://earlham-sherlock.github.io/). One of the core software that Sherlock uses is Docker. Docker is an open and widely used technology to isolate Linux processes and provide them a reproducible, well defined runtime environment independent from the actual operating system (Matthias & Kane 2015). Each element in the Sherlock platform is running inside a separate Docker container. A Docker container is a standard unit of software that packages up all of the necessary code and all the dependencies for them, so the application runs quickly and reliably in an isolated environment on any computer. Container orchestration tools are used when a complex solution (like Sherlock) requires the interconnection of many docker containers distributed among multiple separate Linux machines. In Sherlock, we are using the standard Docker Swarm orchestration platform (Smith 2017), as it is easy to use and much easier to operate than other orchestration frameworks (like Kubernetes (https://kubernetes.io)). These industry standard technologies make the management (starting, stopping and monitoring) of Sherlock easy, while also enabling us to implement more advanced features, for example the on-demand scaling of the analytical cluster where Sherlock is running. This scalability is the biggest advantage of Docker Swarm. The entire cluster can be shut down or reduced to the minimum in the cloud when it is not used, while it can be scaled up to hundreds of nodes if necessary for executing very complex analytical queries. Other key benefits associated with Docker Swarm is the high level of availability offered for applications. This has a hierarchical structure, where several worker nodes and at least one manager node is responsible for handling the worker nodes' resources and ensuring that the cluster operates efficiently.\n\nThe first step for using Sherlock is to start a Docker Swarm with the deployment scripts on a cluster with several worker nodes. These scripts start different services, but each in a separate container. These deployment scripts are configurable ( Figure 1). One can specify exactly how many resources a service can use at once (e.g. CPU, memory allocation). Inside a running Docker Swarm, there are two different main parts of Sherlock: the Hive metastore and the Presto query engine. Sherlock follows the industry best practices in its architecture to enable scalability. Usually, the main idea behind scalable batch processing architectures is the separation of data storage and analytics (Dean & Ghemawat 2008). This architecture allows us to scale the analytical power and the storage size independently from each other and even dynamically. This is the case with Sherlock when deployed in the cloud.\n\nIn this image you can see how the different tools and the deployment scripts are connected to each other inside the core of Sherlock. The blue box represents the Data Lake, where the data is stored. You can see that only the Hive Metastore, the Presto Query Engine and the possible worker nodes have connection to the Data Lake. The box with the dashed line shows the Minio S3 server, where for example, the Sherlock platform can be used to test various features available in Sherlock.\n\nThe Sherlock platform does not include data, instead provides a platform where one can work on and manipulate the data (Figure 1). Then by leveraging powerful database architectures (Presto query engine and Hive metastore) and providing them a channel to connect to the Data Lake (where we are storing our data) the user can run different analytical queries on top of the data files. The starting step for using Sherlock is to submit an SQL query to the Presto query engine, which can answer the biological question in interest and then it will connect to the Data Lake and fetch the necessary data files. Then the final step is to execute the submitted SQL query on top of the data files and then it takes back the results to the user (Figure 1).\n\nThe minimal requirements for Sherlock depend on what the use case is and where Sherlock will be deployed. Sherlock can be used on a single laptop, single virtual machine (VM) or on a distributed cluster. Regarding using Sherlock on a single laptop we recommend using a relatively powerful laptop, which means that it has at least a dual-core processor (CPU) and at least 12 GB memory (RAM), as around half of these resources can be consumed by Sherlock. But the minimum requirement is at least 8 GB RAM and a dual-core processor. On the other hand, if the user wants to use it on a cluster with nodes, we will assume to have at least two machines with 32 GB memory (RAM) for each, and each of them having 8 processor (CPU) cores, but this can be scaled down to 16 GB RAM and 6 CPU cores.\n\nQuery engine\n\nThe first core part of Sherlock is the Query Engine (Figure 2A), which is responsible for running the given question through SQL commands on top of the data files and retrieving the ‘answer’ for the user. A Query Engines, for example Hive, Impala, Spark SQL or Presto, are distributed, mostly stateless and scalable technologies. In our case, we are using the Presto Query Engine (https://prestodb.io), developed by Facebook. Presto provides a high-performance Query Engine wrapped in an easily deployable and maintainable package. Furthermore, the Presto Query Engine can handle many different types of data storage solutions.\n\nA: The structure of Sherlock data platform with the core part, which is the Presto Query Engine. The user can execute different analytical SQL queries with the help of this query engine. Presto will run these queries on top of the data files, which are inside the Data Lake (B) B: The structure of our Data Lake. You can see the four different zones inside the Data Lake, what we are using right now. 1) The raw zone with the raw data from the different external databases. 2) The landing zone, with the Presto compatible text files in JSON Lines format. 3) The master zone, where the data is in a detailed and exact format, called ORC. 4) The project zone, where we store only the data which is needed only for specific projects.\n\nWith Presto, we can formalize analytical questions using SQL. SQL is composed of three main sub-languages: 1) data definition language (DDL), which allows the specification of database schemas; 2) a data manipulation language (DML), which supports operations on data (store, modify, delete; 3) and a data control language (DCL), which enables database administrators to configure security access to databases. On the one hand SQL is designed for working data, which held in a relational database management system (RDBMS), on the other hand it can be used for stream processing in a relational data stream management system (RDSMS). Moreover it is particularly really useful to handle with structured data (Silva et al. 2016). When combined with a Query Engine, SQL can be used to connect to the Data Lake, read and combine the data stored within it and execute different analytical queries, either sequentially or in parallel depending on the power of the given computer.\n\nHive metastore\n\nThe second core part of Sherlock is the Hive metastore (https://docs.cloudera.com/runtime/7.0.1/hive-metastore/topics/hive-hms-introduction.html), which is a service that stores metadata related to Apache Hive and other services, in a backend RDBMS, such as MySQL or PostgreSQL. With regards to Sherlock, Presto stores the metadata about the folders in the Data Lake and the Hive metastore which contains only the meta information about the different folders, which is in the Data Lake. In Sherlock we provide simple scripts to save this metadata in the Data Lake when you want to make a backup or before you want to terminate your analytical cluster.\n\nFor Sherlock, we developed a dockerized version of Presto that also incorporates the Hive metastore. This addition of both the query engine and metastore makes Sherlock cloud-agnostic, so it can be installed at any cloud provider, providing it is a Linux machine with Docker installed. The advantage of running Presto in a dockerized environment is that it is not necessary to install and configure the whole Presto Query Engine manually. Furthermore, it can even be fired up on a local machine, multiple machines or any cloud service as well. On the deployment guide section of the GitHub page of Sherlock (https://earlham-sherlock.github.io/docs/deployment_guide.html), we show how to make a set of Linux virtual machines with Docker installed, then start a distributed Presto cluster by using the Sherlock platform.\n\nA Data Lake is a simple network storage repository that can be accessed by external machines (https://aws.amazon.com/big-data/datalakes-and-analytics/what-is-a-data-lake/). All the data which is imported into, transformed in, or exported from Sherlock will be stored here as simple data files. The biggest advantages of using a Data Lake is that its operation will be the same on a local machine, on the cloud and the data can be stored in a well structured way on a reliable storage. Furthermore it can be scaled independently from the query engine. The technologies in Sherlock are compatible with both of the most common Data Lake solutions; Hadoop Distributed File System (HDFS), which is a distributed file system designed to run on commodity hardware, and with the Simple Storage Service (S3) storage formats. However, we decided to only describe S3 in all of our examples, as S3 is more widely-used, modern, and accessible. Although HDFS is an older standard solution, it has some very powerful features which can result in better performance, but all in all it is also much more difficult to set up, maintain and in most cases its extra features are not necessary for the given project. In contrast, S3 is a standard remote storage API (Application Programming Interface) format, introduced first by Amazon (https://aws.amazon.com/s3/). As of writing, you can purchase S3 storage as a service from all major cloud providers like Digital Ocean, Amazon AWS, Google Cloud or Microsoft Azure and each of these can be compatible with the Sherlock platform.\n\nHaving somewhere to store the data is only one half of having an operational Data Lake. One cannot stress enough how important it is to organize the data in a well defined ‘folder’ structure. Many Data Lake deployments become unusable after a few years due to poor maintenance, resulting in a large amount of data ‘lost’ in the thousands of folders, or inconsistent file formats and no ownership over the data. Our solution is to separate all of the data in the Data Lake into four different main folders, representing different stages of the data and different access patterns in the Data Lake. Inside each of these main folders we can create subfolders, and it is a good practice to incorporate the name of the dataset, the data owner name, the creation date (or other version info), and the file formats somehow into their paths.\n\nWe separated our Data Lake into four different main zones, which are built on top of each other (Figure 2B). The first zone is the raw zone. Into the raw zone, we archived all the database files in their original formats. For example: if we downloaded the human genome, then we put the fasta files here, under a separate subfolder. The name of the subfolder should contain the exact version (for example: hg38_p12) (Figure 3), and also we put a small readme file to the folder, where we listed some metadata, like the date and the url of the download, etc. Usually, these files cannot be opened with Presto, as the file format is incompatible in most of the cases.\n\nThe first part of the folder name is the name of the given database/dataset. In that case the human genome. The next part is the version of the data and the last is the uploaded date to the Data Lake.\n\nThe next zone is the landing zone. We needed to develop specific scripts, called loader scripts, converting and extracting the raw datasets into this zone. We converted the data to a text file in JSON Lines format (https://jsonlines.org), which can be opened by Presto. This is a specific JSON format, where each line of the text file represents a single JSON record. Each JSON file for a given dataset is placed into a separate sub-folder in the landing zone. It is then registered by Presto which sees the dataset now as a table. Then, Presto will be able to load the data, and perform other operations, for example it can execute simple data transformations on the data. However, we do not recommend using the tables in this zone for querying, because processing the large JSON files is very slow.\n\nUsing Presto, we converted the data from the landing zone into an optimized (ordered, indexed, binary) format to the next zone, which is the master zone. The main idea is that we use the tables in the master zone later for analytical queries. Here the data is in a more detailed and exact format, called Optimized Row Columnar (ORC), which is a free and open-source column-oriented data storage format (https://orc.apache.org/docs/). With ORC, Sherlock can perform SQL queries much more faster than using the JSON text file format from the zone below. If necessary, advanced bucketing or partitioning on these tables can be used to optimize the given queries. Furthermore, the master zone contains the ‘single source of truth’. This means that the data here cannot be changed, only extended upon, for example adding a new version of the datasets.\n\nThe last zone is the Project zone. This is where we save the tables that are needed only for specific projects. We can even create multiple project zones, one for each group, project or user. It is important to have a rule to indicate the owner of the tables, as mentioned before this dramatically increases the effectiveness of Sherlock and ease of maintenance.\n\nHere, we describe the key steps of how the query engine and the Data Lake can be used together (Figure 4). The first step is to submit an SQL query to the Presto query engine, then it will connect to the Data Lake and fetch the necessary data files. The third step is to execute the in-memory distributed query and then it takes back the results to the user.\n\nThis image represents the different steps, how a user can submit an analytical SQL query and what is the real path of this query until the results come back to the user.\n\nIt is important to mention that Sherlock has been designed for biologists, especially for network and system biologists. It can contain specific, interaction, expression and genome data-related databases thanks to loader scripts that are able to create the specific file formats from the source databases and upload them into the Data Lake. With these individual loader scripts, users can make and work with the necessary file formats from the different data sources without major time and coding efforts with their own scripts. The whole source code of the platform and the loader scripts ( Table 1) are freely available on Github: https://github.com/earlham-sherlock/earlham-sherlock.github.io\n\nIn the next section, we will outline three different use cases on how Sherlock can be used and what we can use it for.\n\n\nUse Cases\n\nOne of the most crucial and common tasks for those working in the field of bioinformatics is ID mapping. It is difficult to work with many different datasets from many different sources, all carrying diverse identifiers. In addition, these identifiers can have clashing structures, which makes it complicated and time consuming to work with them. Users have to make different scripts or use different tools to work with these identifiers at once. Nowadays the main and key idea behind these ID mapping steps is to have a separate table or tables, called mapping tables, which contain the different identifiers, but only those. The best practice is to have only one mapping table which contains all of the necessary identifiers for a given project. The limitation with this approach is when a team has to work with so many different identifiers at once means that this mapping table can be really large, and it can take a lot of time to go through it and get the data, which is needed.\n\nIn Sherlock, users can work with just one mapping table, which can be made easily with the help of the provided loader scripts from the Github repository, and it can significantly shorten the time needed for ID mapping. Sherlock can execute these queries very quickly despite the large size of the mapping tables, owing to the implemented ORC format. To demonstrate this capability in Sherlock, we search for genes from the STRING database (Szklarczyk et al. 2019), which are expressed only in the brain.\n\nFigure 5 shows three tables. The string_proteins table contains the information from the STRING database (ensembl ID, taxonomy ID and the pubmed ID of the article); the mapping table contains the mapping between the different type of protein identifiers from different sources (ensembl (https://www.ensembl.org/info/genome/stable_ids/index.html) and uniprot (https://www.uniprot.org/help/accession_numbers); and the tissues table includes tissue information about the protein (uniprot identifier, tissue identifier and tissue name) (Figure 5).\n\n\n\nIt shows the three tables where we want to map between. All of the three tables are located in the master zone inside the Data Lake.\n\nThe SQL query will connect the three tables through the matching protein identifiers, selecting only those which are expressed in the brain according to the matching identifiers (light green and purple). The query results in the first two proteins from the string_proteins table (light green color). If the script does not find any match between the ensembl and uniprot IDs, it skips them, like in the case of the third protein in the string_proteins table.\n\nIn this example, we would like to query the top 100 most highly expressed genes in a given tissue (in this case the human colon), and their protein interactors. The limitation with this use case is similar to the previous one: the user has to download the different structured interaction databases from web resources and write scripts or use online tools to work with the data at once. These are also prerequisite steps with the Sherlock platform (having to download the different sources and running preprocessing steps before working with them), but with the provided loader scripts, the user only has to do it once, and it is less time consuming. With Sherlock, the user can easily get this data from multiple interaction tables at once very quickly, thanks to the ORC format with the following SQL query:\n\n\n\nThis query will select the top 100 highly expressed genes from a table imported from the BGee resource (Bastian et al. 2021) to the master zone. The result will be filtered to return only those genes, which are expressed in the colon, and the query will select their protein interactors as well. The results are ordered by the score, so only the colon genes with the top 100 score and their first neighbours will return as the results.\n\nIn the third example, we would like to enrich a network with interaction data from the Data Lake with the help of Sherlock. We have certain proteins of interest shown on Figure 6, and we would like to investigate if there is any relationship between them (find the interactions). We would also like to enrich this network and our proteins with their first neighbours, using an interaction table loaded from the OmniPath (Türei et al.) (https://omnipathdb.org) database.\n\nA: Here you can see how we can find interconnections between our four chosen proteins. B: How can we enrich our network with the first neighbours of the given proteins.\n\nThe SQL query for finding the connections between the proteins (Figure 6A) is the following:\n\n\n\nTo enrich our network with the first neighbours of the given proteins we have to use a different SQL query (Figure 6B), as below:\n\n\n\nOnly a single logical operator changed between the two queries (AND > OR). The reason is, if we want to find the interactions between the proteins, the query has to select only those interactions from the OmniPath table, where the source and the target protein are uniformly included among the proteins we examined. But in the other case, when we want to enrich the network, it is enough to select all of the interactions where either the source or the target proteins are among the proteins of our interest.\n\n\nDiscussion\n\nSherlock provides a new, gap-filling method for computational biologists, who want not only to store, but very easily and quickly convert, query, generate or even share biological data. This novel platform provides a simple, user-friendly interface to work with common and widely used big data technologies, such as Docker or PrestoDB. Thanks to the ORC format that Sherlock uses, users can work with extremely large datasets in a relatively short time that can facilitate any given research project.\n\nSince we made Sherlock, plenty of platforms like Sherlock have appeared worldwide, but none of them was designed specifically to the field of biology. One of them for example is the Qubole platform (https://www.qubole.com). This platform also offers data storage and analytical queries solutions as well, but the Sherlock platform is cheaper. The data storage place and the virtual machines are also needed to be paid, but all of the source code is freely available and can be customized to the user's liking. On the other hand, Sherlock is specifically designed for biologists. It contains specific database loader scripts which they are able to create and upload the specific file formats to the Data Lake. We are constantly upgrading the already included datasets in our Data Lake. We will provide in the Github repository new database loader scripts (to extend interaction and expression data) and different mapping scripts as well. We will also develop different converting scripts to easily handle between Sherlock compatible and other file formats, such as TSV (tab separated value) or CSV (comma separated value).\n\nSherlock has a lot of features, which are the followings: 1) store all datasets in redundant and organized cloud storage, 2) convert all datasets to common, optimized file formats, 3) execute analytical queries on top of data files, 4) share datasets among different teams/projects, 5) generate operational datasets for certain services or collaborators, 6) it is really useful for any groups/teams in the field of computational biology, who has to work with very large datasets for their projects.\n\nIn the future, we would like to include and upload more and more database loader scripts to cover as many interaction and expression databases as possible. Furthermore we are planning to improve our source code, to make more detailed documentation. Right now, the update of the already included databases in the Data Lake is done manually, but we also would like to automate this with a script, which can handle all of the updates at once. Furthermore, we would like to include more common and general – in the field of biology – examples and show it in the repository. Furthermore, we are planning to develop some tutorials on how Sherlock can be used easily and what information is needed for the given projects. We are going to create short online courses and tutorials as well. Our main goal is to disseminate the Sherlock platform widely and we hope it can be useful and great help to the researchers.\n\n\nConclusion\n\nSherlock provides an open-source platform empowering data management, data analytics and collaboration through modern big data technologies. Utilizing the dockerization of Presto and the Hive Metastore, Sherlock is not only powerful but also a flexible and fast solution to effectively and efficiently store and analyze large biological datasets. Sherlock can be used to execute queries on top of a Data Lake, where all the data is stored in a ‘folder-like’ structure providing the added benefit of well defined folder structures also helps and encourages correct data management of biological data. Having a scalable query engine and using ORC format, Sherlock can run SQL queries much faster than other solutions, meaning the user can spend more time working with the data than waiting for a search result, which can significantly reduce the lead time of the research project. In conclusion, Sherlock, through the repurposing concepts and open source tools created by large software companies, provides a ‘plug and play’ state-of-the-art data storage platform for large biological datasets.\n\n\nData availability\n\nAll data underlying the results are available as part of the article and no additional source data are required.\n\n\nSoftware availability\n\nSoftware available from: https://earlham-sherlock.github.io/\n\nSource code available from: https://github.com/earlham-sherlock/earlham-sherlock.github.io\n\nArchived source code available from: http://doi.org/10.5281/zenodo.4738516 (Bohár et al. 2021)\n\nLicense: MIT",
"appendix": "Acknowledgements\n\nWe would like to thank all of the members of the Korcsmaros Group for their advice and feedback.\n\n\nReferences\n\nAshburner M, Ball CA, Blake JA, et al.: Gene Ontology: tool for the unification of biology. Nat. Genet. 2000; 25(1): 25–29. PubMed Abstract | Publisher Full Text | Free Full Text\n\nBohár B, Szalay-Beko M, Fazekas D, et al.: earlham-sherlock/earlham-sherlock.github.io: First release of the official Sherlock platform (Version v1.0.0). Zenodo. 2021, May 5. Publisher Full Text\n\nBastian FB, Roux J, Niknejad A, et al.: The Bgee suite: integrated curated expression atlas and comparative transcriptomics in animals. Nucleic Acids Res. 2021; 49(D1): D831–47. PubMed Abstract | Publisher Full Text | Free Full Text\n\nCalderone A, Castagnoli L, Cesareni G: mentha: a resource for browsing integrated protein-interaction networks. Nat. Methods. 2013; 10(8): 690–91. PubMed Abstract | Publisher Full Text\n\nDas J, Yu H: HINT: High-quality protein interactomes and their applications in understanding human disease. BMC Syst. Biol. 2012; 6: 92. PubMed Abstract | Publisher Full Text | Free Full Text\n\nDean J, Ghemawat S: MapReduce: simplified data processing on large clusters. Commun. ACM. 2008; 51(1): 107.\n\nGarcia-Alonso L, Iorio F, Matchan A, et al.: Transcription factor activities enhance markers of drug sensitivity in cancer. Cancer Res. 2018; 78(3): 769–780. PubMed Abstract | Publisher Full Text | Free Full Text\n\nGreene CS, Tan J, Ung M, et al.: Big data bioinformatics. J. Cell. Physiol. 2014; 229(12): 1896–1900. PubMed Abstract | Publisher Full Text | Free Full Text\n\nHuttlin EL, Bruckner RJ, Navarrete-Perea J, et al.: Dual Proteome-scale Networks Reveal Cell-specific Remodeling of the Human Interactome. BioRxiv. 2020.\n\nKasson PM: Computational biology in the cloud: methods and new insights from computing at scale. Biocomputing 2013. WORLD SCIENTIFIC; 2012, pp. 451–53. PubMed Abstract\n\nKhine PP, Wang ZS: Data lake: a new ideology in big data era. ITM Web of Conferences. 2018: 17: 03025. Publisher Full Text\n\nLi T, Wernersson R, Hansen RB, et al.: A scored human protein-protein interaction network to catalyze genomic interpretation. Nat. Methods. 2017; 14(1): 61–64. PubMed Abstract | Publisher Full Text | Free Full Text\n\nLuck K, Kim D-K, Lambourne L, et al.: A reference map of the human binary protein interactome. Nature. 2020; 580(7803): 402–8. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMarx V: The Big Challenges of Big Data. Nat Methods. 2013. Publisher Full Text\n\nMatthias K, Kane SP: Docker: Up & Running: Shipping Reliable Containers in Production. Sebastopol, CA: O’Reilly Media. 1st ed.2015.\n\nMungall CJ, Torniai C, Gkoutos GV, et al.: Uberon, an integrative multi-species anatomy ontology. Genome Biol. 2012; 13(1): R5. PubMed Abstract | Publisher Full Text | Free Full Text\n\nOrchard S, Ammari M, Aranda B, et al.: The MIntAct project - IntAct as a common curation platform for 11 molecular interaction databases. Nucleic Acids Res. 2014; 42(Database issue): D358–63. PubMed Abstract | Publisher Full Text | Free Full Text\n\nRazick S, Magklaras G, Donaldson IM: iRefIndex: a consolidated protein interaction database with provenance. BMC Bioinformatics. 2008. 9: 405. PubMed Abstract | Publisher Full Text | Free Full Text\n\nSilva YN, Almeida I, Queiroz M: SQL: from traditional databases to big data. Proceedings of the 47th ACM Technical Symposium on Computing Science Education - SIGCSE ’16. New York, New York, USA: ACM Press; 2016; pp. 413–18.\n\nSmigielski EM, Sirotkin K, Ward M, et al.: dbSNP: a database of single nucleotide polymorphisms. Nucleic Acids Res. 2000; 28(1): 352–55. PubMed Abstract | Publisher Full Text | Free Full Text\n\nSmith R: Docker Orchestration. 2017; Packt Publishing\n\nSzklarczyk D, Gable AL, Lyon D, et al.: STRING v11: protein-protein association networks with increased coverage, supporting functional discovery in genome-wide experimental datasets. Nucleic Acids Res. 2019; 47(D1): D607–13. PubMed Abstract | Publisher Full Text | Free Full Text\n\nTürei D, Valdeolivas A, Gul L, et al.: Integrated intra- and intercellular signaling knowledge for multicellular omics analysis. Mol. Syst. Biol. 17(3): e9923. PubMed Abstract | Publisher Full Text | Free Full Text\n\nUniProt Consortium: UniProt: the universal protein knowledgebase in 2021. Nucleic Acids Res. 2021; 49(D1): D480–89. PubMed Abstract | Publisher Full Text | Free Full Text"
}
|
[
{
"id": "85971",
"date": "17 Jun 2021",
"name": "John H. Morris",
"expertise": [
"Reviewer Expertise computational biology"
],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nIn this article, the authors describe a system, Sherlock, for storing and accessing data using SQL. The system described is flexible, and uses modern approaches to the standard ETL (extract, transform, load) processes common in a data warehouse solution. The system seems like it would be extremely useful for a narrow set of use cases, somewhat useful for a larger set of use cases, and of questionable utility for most computational biology use cases. Sherlock's main utility, it seems to me, is for facilities that have large repositories of self-generated data. In that circumstance, the overhead of learning the column names, appropriate foreign keys, and the appropriate SQL syntax to manipulate the data would be well-warranted, and I would imagine in those circumstances, the data could be stored locally on some fast media that would provide significant performance advantages, particularly given the distributed nature of presto. I can also see Sherlock being useful in circumstances where there is a relatively static pipeline that operates on data sources that are more commonly updated. In this circumstance, the ability to have scripts to stage the data and use a common SQL syntax that doesn't change would be useful. In other circumstances, I'm less convinced. First, while I appreciate the value of the multiple-level folder structure (Raw Zone --> Landing Zone --> Master Zone --> Project Zone) that results in a number of copies of databases that could be extremely large. The examples given are relatively small, but consider downloading TrEMBL, which is 132 Gb compressed, and reprocessing that to get at least 3 copies (assuming one would subset it for the Project Zone). The scary part is that TrEMBL is small by modern standards -- image datasets are significantly larger. Second, while I agree that for software developers, SQL is a well-known and convenient query language, it is certainly non-trivial to construct efficient queries and not the best interactive tool for exploring data sets. Further, there is no attempt to fit this tool into the more broad set of tools commonly used by computational biologists. For example, why would I use Sherlock for scRNA-seq analysis if I have access to Galaxy or AnVIL?\nSo, not to be totally negative, I think Sherlock has a place in the tool suite for computational biology, but I don't think that place is well articulated in the article. My recommendations for improving the article are:\nThe second sentence in the introduction talks about working on bespoke datasets, which I suspect is the crux of Sherlock. The rest of the article seems to ignore that, and even the use cases focus purely on public data. This weakens the case for Sherlock. The use cases are trivially (OK, so relatively) easy using a combination of python, perhaps with pandas, data files and web services. The power of Sherlock is the ability to integrate public databases with bespoke databases, but that really isn't highlighted sufficiently. That needs to be the focus on the user cases, I think.\n\nDiscuss Sherlock in the context of other tools (not technologies) that support computational biology, such as Galaxy and AnVIL as two examples. When would I use those tools? When would I use Sherlock? Why is Sherlock better in that case?\n\nBe clearer as to the target user. In several cases, the article states that this is \"designed specifically for biologists\". I actually don't think that's correct. It's designed for computational biologists or bioinformaticians with a very strong computational background. I don't know many bench biologists who would be able to formulate some of the SQL queries, even if it was easy to figure out all of the column names. I don't *think* the authors believe that SQL is the user interface for the system as much as an API for accessing the data, but that also wasn't clear in the text.\n\nThe role of Hive is somewhat obscure to me. I got that it stores the metadata, but *what* metadata? Is that where I can look up the schema to get the column names for joins and projections? In the use cases, it seems like there were several jumps in logic. Where does the user figure out that the ensemble ID in the STRING database is stored as \"ens_id\"? How well exposed is the schema for each of the tables in the data lake and how does a user (who didn't happen to be the one who imported the data) figure that out?\n\nOne of the main points in the article is the potential for high performance due to the parallelism of presto, but there were no performance numbers offered, either for the ETL phase or for the queries themselves.\n\nFinally, as a more minor point, there are several places where the manuscript could use a bit of an edit pass. For example, on page 2 it states \"A Query Engines, for example \" shouldn't be plural.\n\nSherlock clearly represents a *lot* of work, and I do believe that it could be valuable to the appropriate audience and that it should be published. My take on the manuscript is that it's not clear who it is written for. If it's written for a computer scientist, then information about performance metrics and local vs. cloud storage trade-offs and scalability, etc., are missing. If it's written for the computational biologist, then it's not really necessary to address the trade-offs between docker swarm and kubernetes, or why SQL can be considered composed of three main sub-languages, but it is important to explain why Sherlock is better than Galaxy or AnVIL and a little more detail on how the user figures out things like column names, etc.\nSorry if this comes across as overly negative. I honestly do see how much work is involved and I can imagine where it might be extremely useful in some environments, but that just doesn't come across clearly in this submission.\n\nIs the rationale for developing the new software tool clearly explained? Partly\n\nIs the description of the software tool technically sound? Yes\n\nAre sufficient details of the code, methods and analysis (if applicable) provided to allow replication of the software development and its use by others? Yes\n\nIs sufficient information provided to allow interpretation of the expected output datasets and any results generated using the tool? Yes\n\nAre the conclusions about the tool and its performance adequately supported by the findings presented in the article? Partly",
"responses": [
{
"c_id": "8552",
"date": "10 Aug 2022",
"name": "Tamás Korcsmáros",
"role": "Author Response",
"response": "Response to Reviewers for Manuscript ID: f1000research.52791.1 We thank the reviewers for their feedback on our manuscript and the constructive comments. The comments and questions led to a significant and appropriate revision of the manuscript. Answers for reviewer John H. Morris In this article, the authors describe a system, Sherlock, for storing and accessing data using SQL. The system described is flexible, and uses modern approaches to the standard ETL (extract, transform, load) processes common in a data warehouse solution. The system seems like it would be extremely useful for a narrow set of use cases, somewhat useful for a larger set of use cases, and of questionable utility for most computational biology use cases. Sherlock's main utility, it seems to me, is for facilities that have large repositories of self-generated data. In that circumstance, the overhead of learning the column names, appropriate foreign keys, and the appropriate SQL syntax to manipulate the data would be well-warranted, and I would imagine in those circumstances, the data could be stored locally on some fast media that would provide significant performance advantages, particularly given the distributed nature of presto. I can also see Sherlock being useful in circumstances where there is a relatively static pipeline that operates on data sources that are more commonly updated. In this circumstance, the ability to have scripts to stage the data and use a common SQL syntax that doesn't change would be useful. We thank the Reviewer for these general comments and we agree with the specific use cases described by the Reviewer on where Sherlock could be useful. In other circumstances, I'm less convinced. First, while I appreciate the value of the multiple-level folder structure (Raw Zone --> Landing Zone --> Master Zone --> Project Zone) that results in a number of copies of databases that could be extremely large. The examples given are relatively small, but consider downloading TrEMBL, which is 132 Gb compressed, and reprocessing that to get at least 3 copies (assuming one would subset it for the Project Zone). The scary part is that TrEMBL is small by modern standards -- image datasets are significantly larger. Second, while I agree that for software developers, SQL is a well-known and convenient query language, it is certainly non-trivial to construct efficient queries and not the best interactive tool for exploring data sets. Further, there is no attempt to fit this tool into the more broad set of tools commonly used by computational biologists. For example, why would I use Sherlock for scRNA-seq analysis if I have access to Galaxy or AnVIL? We thank the Reviewer for this comment, which is really helpful as these were not properly covered in the original submission. Regarding the multi-level folder structure, we added a paragraph into the end of “The Data Lake - data storage” section to explain its usefulness further. Regarding the last part of this comment, we added paragraphs to the “Discussion” section of the revised manuscript. So, not to be totally negative, I think Sherlock has a place in the tool suite for computational biology, but I don't think that place is well articulated in the article. My recommendations for improving the article are: The second sentence in the introduction talks about working on bespoke datasets, which I suspect is the crux of Sherlock. The rest of the article seems to ignore that, and even the use cases focus purely on public data. This weakens the case for Sherlock. The use cases are trivially (OK, so relatively) easy using a combination of python, perhaps with pandas, data files and web services. The power of Sherlock is the ability to integrate public databases with bespoke databases, but that really isn't highlighted sufficiently. That needs to be the focus on the user cases, I think. We would like to thank you for this comment and also thanks for the suggestions, we modified the manuscript accordingly. We added that part into the “Overview of the platform” section. Discuss Sherlock in the context of other tools (not technologies) that support computational biology, such as Galaxy and AnVIL as two examples. When would I use those tools? When would I use Sherlock? Why is Sherlock better in that case? Thank you for these important questions not yet addressed in the manuscript. We added new paragraphs about AnViL and Galaxy into the “Discussion” section to address these. Be clearer as to the target user. In several cases, the article states that this is \"designed specifically for biologists\". I actually don't think that's correct. It's designed for computational biologists or bioinformaticians with a very strong computational background. I don't know many bench biologists who would be able to formulate some of the SQL queries, even if it was easy to figure out all of the column names. I don't *think* the authors believe that SQL is the user interface for the system as much as an API for accessing the data, but that also wasn't clear in the text. Thank you for this comment as well. This is a valid point as indeed Sherlock was designed for computational biologists or data scientists. Therefore, we clarified it in the revised manuscript and updated the title as well. The role of Hive is somewhat obscure to me. I got that it stores the metadata, but *what* metadata? Is that where I can look up the schema to get the column names for joins and projections? In the use cases, it seems like there were several jumps in logic. Where does the user figure out that the ensemble ID in the STRING database is stored as \"ens_id\"? How well exposed is the schema for each of the tables in the data lake and how does a user (who didn't happen to be the one who imported the data) figure that out? Thank you for raising this. Hive is actually more of a technical part, which is used by PrestoDB. Sherlock stores metadata (columns structure) about the tables for Presto to query the files. For best practice, each user/user group needs to add a schema to describe what is inside the tables (wiki page, loader scripts, SQL editors). We have extended the manuscript to clarify these points in the “Functionality of Sherlock” section. One of the main points in the article is the potential for high performance due to the parallelism of presto, but there were no performance numbers offered, either for the ETL phase or for the queries themselves. Thank you for pointing out that this comparison was missing. We added test measurement to the manuscript into the “The Data Lake - data storage” section. Finally, as a more minor point, there are several places where the manuscript could use a bit of an edit pass. For example, on page 2 it states \"A Query Engines, for example \" shouldn't be plural. Thank you for raising this, we reviewed and corrected the text now. Sherlock clearly represents a *lot* of work, and I do believe that it could be valuable to the appropriate audience and that it should be published. My take on the manuscript is that it's not clear who it is written for. If it's written for a computer scientist, then information about performance metrics and local vs. cloud storage trade-offs and scalability, etc., are missing. If it's written for the computational biologist, then it's not really necessary to address the trade-offs between docker swarm and kubernetes, or why SQL can be considered composed of three main sub-languages, but it is important to explain why Sherlock is better than Galaxy or AnVIL and a little more detail on how the user figures out things like column names, etc. Thank you very much for this summary and the suggestions. We agree with the Reviewer's view. Accordingly, we modified the manuscript throughout to meet with the interest and needs of a computational biologist. In some cases, we kept some background information (such as trade-offs between docker swarm and kubernetes) but rephrased them in a restructured arrangement. Sorry if this comes across as overly negative. I honestly do see how much work is involved and I can imagine where it might be extremely useful in some environments, but that just doesn't come across clearly in this submission. Thank you very much for all of the constructive criticism and helping us to improve the manuscript."
}
]
},
{
"id": "85970",
"date": "29 Jun 2021",
"name": "Nadezhda Doncheva",
"expertise": [
"Reviewer Expertise Computational and network biology",
"development of software tools",
"analysis and integration of large datasets"
],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nIn this article, the authors present an open-source platform called Sherlock, which can be very useful for computational biologists working with a lot of different datasets within one or several projects. The platform is designed to make use of well-known resources and concepts such as Docker, and PrestoDB. Sherlock is easily extendable and can be used both on a local computer or in connection with a cloud storage solution. Once Sherlock is properly set up and the needed data sets imported and formatted, the data can be integrated and analyzed using SQL queries as shown in the three simple use cases.\n\nSherlock can become a great addition to the suite of tools used by computational biologists, especially when it comes to bigger projects involving a lot of public datasets as well as own data generated by collaborators. The article is nicely written and gives a good overview of the Sherlock platform as well as possible use cases. However, it can be further improved in three main aspects as detailed below: making clear who is the target audience, improving the documentation about setting up Sherlock, and improving the flow of the Implementation and Operation section.\nBoth the title and abstract mention that Sherlock is designed for biologists and only on page 6, the text mentions computational biologists specifically. However, both from the description of the setup and the specific use cases, it seems to me that the actual target audience should be clearly stated as “computational biologists”. It is worth considering changing the “for biology” part in the title since it is not very clear or specific as it is now.\n\nOne major concern is that neither the manuscript nor the online documentation seem to describe in enough detail how to set up the data needed for the three presented use cases. There is a nice deployment guide and the use case examples seem reasonable, but the step in-between is missing. The use cases assume that the user has an already configured Sherlock system with the necessary files stored, properly formatted and the right tables created, etc. I specifically checked the GitHub documentation, among others the “Loading … data to Sherlock” sections and I don’t think enough detail is given for a user like me to set it up (and I consider myself a computational biologist). I would need to know which raw data files from STRING were retrieved and stored in the raw zone, how were the JSON files generated that need to be copied to the landing zone, etc. I was able to find the scripts, which should help for this step (https://github.com/earlham-sherlock/earlham-sherlock.github.io/tree/master/loaders) but they are also not described in enough detail, especially in connection with the use cases. So, what I think is needed is the following:\nadd a short description to each use case in the manuscript about which databases are needed to execute the example queries;\n\non GitHub, add a step-by-step tutorial for how to set up the specific databases/tables needed for the use cases. For use case 1, the tutorial should include information about how and from what data to create the tables “master.mapping”, ”master.tissues”, ”master.string_proteins”, while for use case 2 and 3, the needed tables are “master.bgee_2020_11_16” and “master.omnipath_2020_10_04”.\n\nAnother major comment is that the structure of the whole section “Implementation and Operation” can benefit from a bit of reorganization and section renaming. In the following, I will give some specific details and examples of how this can be done.\nThe Overview section begins with a lengthy description of Docker and Docker Swarm and only gives an overview of the different components of Sherlock at the end of the section. The section will read much better if it begins with a high-level overview of the different components as it is partly done in the third paragraph (last one on page 3). This part can also refer to Figure 1. Then, each of the components can be explained in more or less detail (depending on the order and what follows in the next sections). For example, the end of paragraph one, which describes the scalability of Docker Swarm, also fits well with the last paragraph of the Overview section. This section could also be renamed to “Overview of the Sherlock platform”.\n\nIn the Overview section, there seems to be a bit of confusion about the architecture & setup versus the usage workflow. The second paragraph starts with a sentence stating that “the first step of using Sherlock is to start a Docker Swarm”, while the third paragraph states that “the starting step for using Sherlock is to submit an SQL query to the Presto query engine”. I can see how both of these statements are true, but it will be easier to read if it is explained that one thing is part of the general, one-time setup of the system (which also fits more with the whole architecture), while the other one is the actual user workflow in a day-to-day usage (more relevant to the “Functionality of Sherlock” section).\n\nAs a follow-up on that, it would be possible to move the whole description of Docker together with the “Query engine” and “Hive metastore” subsections into a section entitled “Architecture” or optionally “Architecture and setup” (instead of “Sherlock as a platform”).\n\nThe last section about the functionality of Sherlock is currently very short and actually describes this day-to-day workflow. It could easily become part of the first overview section or else be extended by a few more details that do not fit so well into the Overview/Architecture sections.\n\nSo, for the whole big section, the order would be “Overview of the Sherlock platform”, “Architecture (and setup)”, “Deployment”, “The Data lake – data storage” and optionally “Functionality of Sherlock”.\n\nIn the following list, I have pointed out several minor things about the text or figures that can also be improved.\nThe abstract says that “Sherlock is designed to analyse, process, query and extract the information from extremely complex and large datasets.” I think to be precise, it should say that it is designed “to facilitate/enable/allow users in analysing, processing, … “\n\nThe description of Docker in the first paragraph of the Overview section could be shortened a bit (or moved to a more fitting place a bit later in the description).\n\nThe legend of Figure 1 can be improved by describing the elements form left to right instead of right to left. It is not clear what a “Minio S3 server” is since it is not mentioned anywhere else in the manuscript. The first column of boxes in the Figure could also be surrounded by a bigger box and referred to as the user input (configuration) or something similar. Then it would be easier to refer to it.\n\nPage 4, second paragraph: “A Query Engines… are distributed” should be either singular or plural but not both.\n\nPage 4, third paragraph: the phrase “designed for working data” should probably be “designed for working with data” and “which held in” should be “which is held in”.\n\nPage 4, third paragraph: the sentence “Moreover it is particularly really useful to handle with structured data” can be rephrased for more clarity.\n\nPage 4, last paragraph: the sentence “With regards to Sherlock, Presto stores the metadata about the folders in the Data Lake and Hive metastore, which contains only the meta information about the different folders, which is in the Data Lake” also needs to be rephrased. There are too many “which” and it is not clear what refers to what and what information is actually contained/known to Presto and Hive metastore.\n\nFigure 2 does not look very nice in the PDF as opposed to the online version on GitHub, which has nicer formatting and quality. I would recommend replacing the figure in the PDF with the online version and making sure it still looks fine.\n\nPage 5, last paragraph: “on a local machine, on the cloud and the data…” should probably be “on a local machine and on the cloud and that the data…”.\n\nPage 5, last paragraph: the semicolon in the sentence “the most common Data Lake solutions;” could be replaced by a colon and “and with the Simple…” by “and Simple …”.\n\nPage 6, first paragraph of “Functionality of Sherlock”: how should the last sentence be understood? Does the user execute a “in-memory distributed query” or is this done also by the Presto engine?\n\nPage 6, last paragraph: the comma in “contain specific, interaction, …” is unnecessary.\n\nPage 7, there is an issue with the Omnipath reference in the table, it looks different and leads to a log-in page in the online version of the article.\n\nPage 7, first sentence “…we will outline three different use cases on how Sherlock can be used and what we can use it for” could be rephrased to sound better. Maybe just “… we will outline three different use cases of Sherlock”.\n\nPage 9, second paragraph: should this sentence “to enrich a network with interaction data” rather say “to retrieve interaction data for a gene list”?\n\nFigure 6: The two actions “Enrich with interconnections” and “Enrich with first neighbors” are repeated. I think it is enough to state this once below the arrow since it nicely describes the actual process. The picture before and after the arrow could have some other labels, but it is also fine if they do not have their own label.\n\nPage 10, second paragraph: the sentence “it contains specific database loader scripts which they are able to create and upload specific file formats to the Data Lake” needs some revising to make clear who is “they”. Maybe it should say “which the users are able to use to upload or can create themselves…”.\n\nPage 10, third paragraph: “followings” should be “following”.\n\nPage 10, last paragraph: there is probably a stop (sentence end) missing between “structure” and “providing” in “a ‘folder-like’ structure providing the added…”.\n\nI am not able to see the images in the html version of the article in Firefox on Mac, I can only see a name (f1db3fb0-55ef-43b8-9ea8-7f6486a79c9a_figure1.gif). If I right-click and try to see it in another tab, I see some XML code that says Access denied. I checked and I don’t have the same issue with other articles.\n\nIs the rationale for developing the new software tool clearly explained? Partly\n\nIs the description of the software tool technically sound? Yes\n\nAre sufficient details of the code, methods and analysis (if applicable) provided to allow replication of the software development and its use by others? Partly\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": "8553",
"date": "10 Aug 2022",
"name": "Tamás Korcsmáros",
"role": "Author Response",
"response": "Response to Reviewers for Manuscript ID: f1000research.52791.1 We thank the reviewers for their feedback on our manuscript and the constructive comments. The comments and questions led to a significant and appropriate revision of the manuscript. Answers for reviewer Nadezhda T. Doncheva In this article, the authors present an open-source platform called Sherlock, which can be very useful for computational biologists working with a lot of different datasets within one or several projects. The platform is designed to make use of well-known resources and concepts such as Docker, and PrestoDB. Sherlock is easily extendable and can be used both on a local computer or in connection with a cloud storage solution. Once Sherlock is properly set up and the needed data sets imported and formatted, the data can be integrated and analyzed using SQL queries as shown in the three simple use cases. Sherlock can become a great addition to the suite of tools used by computational biologists, especially when it comes to bigger projects involving a lot of public datasets as well as own data generated by collaborators. The article is nicely written and gives a good overview of the Sherlock platform as well as possible use cases. However, it can be further improved in three main aspects as detailed below: making clear who is the target audience, improving the documentation about setting up Sherlock, and improving the flow of the Implementation and Operation section. We thank the Reviewer for these general comments and the summary about the manuscript. We also really appreciate the suggestions about the three main aspects. Both the title and abstract mention that Sherlock is designed for biologists and only on page 6, the text mentions computational biologists specifically. However, both from the description of the setup and the specific use cases, it seems to me that the actual target audience should be clearly stated as “computational biologists”. It is worth considering changing the “for biology” part in the title since it is not very clear or specific as it is now. Thank you very much for raising it, it is a good point. We agree with the Reviewer's view, and modified the manuscript throughout to reflect that Sherlock is primarily for computational biologists. One major concern is that neither the manuscript nor the online documentation seem to describe in enough detail how to set up the data needed for the three presented use cases. There is a nice deployment guide and the use case examples seem reasonable, but the step in-between is missing. The use cases assume that the user has an already configured Sherlock system with the necessary files stored, properly formatted and the right tables created, etc. I specifically checked the GitHub documentation, among others the “Loading … data to Sherlock” sections and I don’t think enough detail is given for a user like me to set it up (and I consider myself a computational biologist). I would need to know which raw data files from STRING were retrieved and stored in the raw zone, how were the JSON files generated that need to be copied to the landing zone, etc. I was able to find the scripts, which should help for this step (https://github.com/earlham-sherlock/earlham-sherlock.github.io/tree/master/loaders) but they are also not described in enough detail, especially in connection with the use cases. So, what I think is needed is the following: add a short description to each use case in the manuscript about which databases are needed to execute the example queries. On GitHub, add a step-by-step tutorial for how to set up the specific databases/tables needed for the use cases. For use case 1, the tutorial should include information about how and from what data to create the tables “master.mapping”, ”master.tissues”, ”master.string_proteins”, while for use case 2 and 3, the needed tables are “master.bgee_2020_11_16” and “master.omnipath_2020_10_04”. Thank you very much for your advice, these are really good and useful points. We have added the suggested clarifications for the github repository and for the Use Cases section in the revised manuscript as well. Another major comment is that the structure of the whole section “Implementation and Operation” can benefit from a bit of reorganization and section renaming. In the following, I will give some specific details and examples of how this can be done. The Overview section begins with a lengthy description of Docker and Docker Swarm and only gives an overview of the different components of Sherlock at the end of the section. The section will read much better if it begins with a high-level overview of the different components as it is partly done in the third paragraph (last one on page 3). This part can also refer to Figure 1. Then, each of the components can be explained in more or less detail (depending on the order and what follows in the next sections). For example, the end of paragraph one, which describes the scalability of Docker Swarm, also fits well with the last paragraph of the Overview section. This section could also be renamed to “Overview of the Sherlock platform”. Thank you for bringing this to our attention, we have now improved this section and made the suggested restructuring changes. In the Overview section, there seems to be a bit of confusion about the architecture & setup versus the usage workflow. The second paragraph starts with a sentence stating that “the first step of using Sherlock is to start a Docker Swarm”, while the third paragraph states that “the starting step for using Sherlock is to submit an SQL query to the Presto query engine”. I can see how both of these statements are true, but it will be easier to read if it is explained that one thing is part of the general, one-time setup of the system (which also fits more with the whole architecture), while the other one is the actual user workflow in a day-to-day usage (more relevant to the “Functionality of Sherlock” section). Thank you for raising it as well, we have fixed these inaccuracies. In the Use Cases section, we added a general set up guide of the platform and the Data Lake. After that, we introduce how to run basic analytical SQL queries in different use cases. As a follow-up on that, it would be possible to move the whole description of Docker together with the “Query engine” and “Hive metastore” subsections into a section entitled “Architecture” or optionally “Architecture and setup” (instead of “Sherlock as a platform”). Thank you for your suggestion. We changed the section names and structure accordingly. The last section about the functionality of Sherlock is currently very short and actually describes this day-to-day workflow. It could easily become part of the first overview section or else be extended by a few more details that do not fit so well into the Overview/Architecture sections. Thank you, this is a good idea. We created a new subsection for the whole docker part and also extended it with more details. So, for the whole big section, the order would be “Overview of the Sherlock platform”, “Architecture (and setup)”, “Deployment”, “The Data lake – data storage” and optionally “Functionality of Sherlock”. Thank you for your advice. We have restructured the whole Implementation and Operation section based on your suggestions, and also we fixed the minor points that you raised. Thank you very much for all of the constructive comments that helped us to improve the manuscript."
}
]
}
] | 1
|
https://f1000research.com/articles/10-409
|
https://f1000research.com/articles/12-45/v1
|
11 Jan 23
|
{
"type": "Research Article",
"title": "At the nexus of ludology and narratology: Advances in reality-based story-driven games",
"authors": [
"Lei Chen",
"David Dowling",
"Christopher Goetz",
"David Dowling",
"Christopher Goetz"
],
"abstract": "Story-driven games are growing in popularity across a wide range of genres. However, the narrative potential of video games is still being debated, particularly in light of the so-called tension between gameplay and storytelling. This study proposes that rules and game mechanics perform narrative semiotic functions, offering a ludic grammar of interactive storytelling. Case studies of four representative games show through exploratory player action shaped by rules, the medium of video games can generate meanings that traditional media cannot, thereby better achieving their narrative goals.",
"keywords": [
"Storytelling",
"video games",
"ludology",
"narratology",
"gameplay",
"Detroit: Become Human",
"Chinese Parents"
],
"content": "Introduction\n\nThe last decade has seen a surge of creative experimentation in video game storytelling granting unprecedented agency to audiences of reality-based storytelling. Digital games increasingly combine cinematic perceptual realism with a modular flexibility and immediacy that exceeds even gamebooks as a medium. Although Detroit: Become Human (2018) has generated a great deal of popular online commentary as well as scholarly interest, the assumption that visually immersive story worlds hold the key to narrative power in the space of video games has been effectively dispelled, especially in the cases of non-action, hand-illustrated text box games Closed Hands (2021); Bury Me, My Love (2017); and Chinese Parents (2018).\n\nHowever, in the opinion of some researchers, such as Ian Bogost's, video games have still not proven themselves to be a successful storytelling medium, since even if games can tell stories well, film, television, and literature all tell them better (Bogost 2017). As Bogost (2017) points out, stories told by walking simulator games, like What Remains of Edith Finch, can also be experienced as a traditional time-based narrative, such as movie, since they are entirely linear, and interacting with the environment only gets in the way. When players often cannot find the location of the next scene that contains narrative content. In his view, rather than attempting to tell stories capable of the narrative power associated with cinema and literature, video games are more adept at generating meaning through player actions, particularly by offering independent exploration of virtual worlds and systems within a defined set of rules (Bogost 2017).\n\nThe purpose of this paper is to reveal that while it is true that poorly designed games can cause game mechanics to hinder the story experience, the tension between the two is not inevitable. In fact, game mechanics are essentially symbiotic with narrative. We demonstrate precisely how ludic elements, such as rules, interfaces, and technology of the text box, have become the core game mechanic sparking the latest advances in video game storytelling.\n\nAnother essential argument this essay pursues in each case study is that the supposed opposition between ludology and narratology proves illusory, especially when play itself is figured as an act of reading. Reading a game is a form of immersion in a reality-based character’s situation that demands critical understanding and interpretation. While choices in some games can be made haphazardly or even at random, making an informed choice involves players in a series of expected and unexpected outcomes. Informed choices grow in significance as consequences accumulate in what amounts to the co-construction of the game’s narrative and its meanings. We suggest that the rich possibilities of meaning are most interesting in nonfiction games, which explicitly address and seek to comment upon explicit, real-world phenomena.\n\nThus, the four games examined in this study are reality-based. Their stories engage seriously with social, political, and psychological topics. Namely the non-fiction topics informing these case studies are American slavery (Detroit: Become Human), the Syrian refugee crisis (Bury Me, My Love), the 2017 Manchester, England terrorist bombing (Closed Hands), and the collective memory of childrearing in China during the 1980s and 1990s (Chinese Parents). These games combine earnest and intimate engagement with political topics of global concern with especially ambitious and complex writing. Further, these games are all developed outside the U.S.—the first two are French, followed by British and Chinese titles—highlights the global reach of video game storytelling. Our reading of these games interrogates the apparent correlation between the ethical treatment of reality-based topics and interactive storytelling as a game mechanic. The broader contexts of these games cast questions about player agency (and choice) in a new light.\n\n\nTheorizing ludic narrativity\n\nOur study builds on Green’s (2017) revelation that “sharp divides across ludological and narratological lines must be erased, for the larger benefit of both avenues of video game exploration” (14).\n\nAlthough Frasca (2003) has claimed that the ludologist vs. narratologist debate never existed since “ludologists love stories too,” his earlier work proposes the dichotomy of simulation versus narrative and suggests that the true prowess of the medium of the video game lies in its performance of the former. Frasca (2001) indeed is responsible for contributing to the early definition of ludology as the study of games, which he defined in opposition to literary approaches, underscoring that “games cannot be understood through theories derived from narrative.” Ludology thus came to be understood in the early 2000s as the study of gameplay and game structure as defined by rules and restrictions for player interaction. Juul (2001) adds that narratives are often confused with video games because the player can retell the story of their game session so that narrative sequence (like the reward of a cut scene) can be generated through (yet is not equivalent with) gameplay. Several structural traits are shared by video games and narratives. Crucially, ludologists and narratologists may be operating according to disparate definitions of narrative, with the former understanding storytelling as a fixed causal chain of events that are recounted and/or reenacted, whereas the latter sees narrative “as a loose fictional frame” applied to a variety of experiential phenomena (Egenfeldt-Nielsen et al. 2020, 226; Ryan 2004; Grodal 2003). Early thinking in ludology views video games as a “story-generating systems” akin to real life—that is, as “primary, real-time phenomena” (Aarseth 2004, 50). Narrative, on the other hand, is seen as a “secondary” phenomenon, as “a revision of the primary event” (50). Such a view positions games alongside “primary,” real-world phenomena, and seems to automatically foreclose the possibility of a strong secondary relationship (e.g., between a game and a serious, real-world issue that it comments upon). The early ludology approach thus seems more appropriate for games with fictional frames and interests that seem to swerve away from reality and are instead rooted in fantasy and empowerment. In contrast, the games at the heart of this study are understood as profoundly related to the real-world issues they explore. And the text box itself—emblematic of narrative in games more generally—serves as both a mechanical reworking of real-world issues (their organization into key decision point, as a perhaps “secondary” framing of a primary event) and as the point of entry for the player’s own significant involvement in these issues.\n\nNotably, the ethics narratives of our cases represent the digital remediation of print culture’s Choose Your Own Adventure stories first published by Chooseco (which sued Netflix for infringing upon its trademark in the aforementioned interactive film Bandersnatch) in the 1970s. Crucially, the Choose Your Own Adventure gamebooks declined in popularity with the rise of narrative video games from the late 1980s through the 1990s. As Jamison (2022) notes, by the mid-1990s, the novel experience of controlling the narrative you are inside of “felt more seductive in digital media—where the worlds were more visually immersive, and the choices more constant.” Choices in video games also encompass a much wider range of narrative affordances and actions, from the inconsequential to the existentially significant, than printed gamebooks. The books were discontinued in 1999, due in large part to the capacity of video games to tell this type of story in a much more powerful register, one capable of addressing enduring political and social questions in serious, reality-based areas such as race and immigration. Janet Murray (1997) famously ponders who will be the first Shakespeare of the medium, a “cyberbard” capable of bending the technology to more artistic effects (71). Given the evolutionary trajectory of the medium, however, we are more likely to encounter a critical historian like Thucydides than a Shakespeare. The master storytellers of the medium have begun to emerge not in the realm of escapist fictional content, but in reality-based video games on major social and political topics, narratives that unfold on epic scales with all the rhetorical techniques of fiction. Nonfictional subjects previously reserved for historians and journalists are now in the hands of game developers.\n\nThis movement in game development breaks down the binary between ludology and narratology anticipated by Frasca’s (2003) own recanting of his previously firmer stance to counter accusations of defensiveness against the so-called “foreign” colonization of game studies by literary scholars on the one hand, and a strain of essentialist radicalism on the other. He notes that “the work of the so-called ludologists does not reject narrative, nor does it want to finish narrative elements in video games,” as he is careful to point out that “accusations of radicalization of this debate are totally unfounded.” Our approach instead seeks to show empirical evidence through the games themselves and their mechanics for storytelling that the terms of the debate are unnecessarily locked into a false binary. Seeking overlaps in the two camps through the oblique concessions of ludologists to narrativity, may provide a starting point, but is essentially a misplaced endeavor that elides the need for discussion of actual games and how they function to produce stories.\n\nOur approach thus resonates with Green’s (2017) point that privileging ludology over narrative inappropriately “reduces video game study to an “either/or” scenario, such that one either considers only the mechanics of a game or examines story in the absence of acknowledging the confluence of technology, game, and player” (13). For games without a clear ending that decides a winner, narrative is not rendered subordinate to skill grinding to attain points, levels and badges. A less acquisitive model, such as that showcased in Walden: A Game (2017), is indeed the intent for Detroit: Become Human. The player’s quest in each game is for the extraction of meaning through interaction with the virtual world. It should be noted that, whereas ludologists such as Bogost (2017) would categorize Walden: A Game as a “walking simulator” that “devolves into conceptualism,” Detroit: Become Human showcases action scenes featuring police conflicts and dramatic hostage situations within a science fictional setting more common to video games. Detroit: Become Human indeed bears all the trappings of a first-person shooter dystopian action game, only with a choice-driven game mechanic in place of combat as the chief mode of gameplay. The humble and underappreciated text box, along with associated devices of the dialogue wheel, and interactive fiction, constitutes the engine behind not only the storytelling prowess of Cage’s magnum opus, but a wave of indie PC games that transform the Choose Your Own Adventure story into a visceral, dynamic form of play fraught with risk, consequence, and temporal pressure. In the cases selected for this study, winning in the traditional sense is not the goal; pursuing a viable life course through a set of existentially important decisions with radically different outcomes instead transforms the player into an author in games granting agency. In non-choice games with less agency, prescribed narrative is performed according to a more linear, cinematic model. All of the cases examined here represent the ways in which the development of branching narratives entails a labor intensive creative process that breathes new life into this still-emerging medium. Building branching narratives in digital games is “an often underappreciated effort,” according to the developer of the PC game Zenuel: “You wouldn’t expect text in a game to be so divisive, or so complex, but when done right, it empowers every area of a game, enriches even the smallest details and makes a world feel like it knows you’re there” (Nelson 2018).\n\nThis form of gameplay mechanic, which we call ludic narrativity, is not to be confused with another type of game featuring unstructured play in an open world, or sandbox. The narrative form associated with sandbox games has been described as the complete graph structure, which grants the player total freedom to navigate along bidirectional paths with all nodes linking to each other (Ryan 2001, 247). A coherent narrative is impossible to guarantee according to this form since the player has full control of action and movement. Instead, in our cases the player follows a series of prompts with multiple outcomes, whereby they navigate their own course through a variety of branching paths. Our cases represent a range of player agency directly correlated to the number of available paths toward different outcomes. Bury Me, My Love offers the least amount of player agency, yet as we explain in its case study, the unilinear, chronological progression of the game—a form akin to sightseeing with a tour guide—is complicated by its multiple distinct vectors with brief side branches, each of which effectively showcases the weight of its real-world subject of the Syrian refugee crisis.\n\nScholarly discussion of narrative in video games has addressed the problem of allowing players to act freely, while ensuring their actions produce an interesting story. Management of this tension is a key reason for the larger scale of interactive writing, as seen in the roughly 130,000 words that constitute the narrative architecture of Closed Hands, a volume far greater than most novels. This vast canvas for storytelling is an extension of expansive digital age storytelling particularly evident with the advent of streaming video on demand (SVOD), that gave rise to binge-watching, and now with podcasting, binge-listening, as mainstream forms of media consumption. The sheer volume of content speaks to the encyclopedic property of interactive storytelling, which is enabled by the data storage capacity of computers, as Murray (1997) observes in Hamlet on the Holodeck, her foundational study highlighting the literary potential for storytelling through emerging technology. Endowed with such a massive capacity for data compared to traditional media forms designed to deliver narratives, spatial storytelling is enabled by the power of computers to represent navigable 3D space. With encyclopedic properties and spatial storytelling, video games can offer either a narrow or wide range of choices to players, depending on the developer’s aims.\n\nIf agency is relatively limited to a narrow range of choices with little impact on the shape of the narrative, the game’s storytelling becomes more performative, as in the case of Bury Me, My Love. Although the game contains 20 endings, the player has little agency in determining the course of each of those narrative lines. Indeed, some players of Bury Me, My Love complained of not having enough agency, showing that the game can be about performance rather than a choice structure. Nonetheless, the ludic quality of each of those narrative threads, as the case study will later demonstrate, suggests that narrativity is the primary game mechanic, although more like advancing pages in a novel (through the game’s simulated WhatsApp interface) rather than steering the outcome of a Hollywood screenplay. Micro choices in dialogue still funnel back to the main channel of the narrative, holding players to the course of the fixed outcome, a kind of virtual jungle cruise with a few minor diversions along the way, but never enough to deviate from the inevitable flow of experience. Murray’s (1997) concept of interactive storytelling posits far greater agency than this game affords, one that envisions an elaborate choice structure like those of Detroit: Become Human, Closed Hands, and Chinese Parents. The participatory properties integral to interactive storytelling are defined as the digital medium’s technological capacity to influence the production of the player’s behavior when engaged with the game. Procedural properties constitute the game’s capacity to execute rule in succession to generate behavior (Murray 1997).\n\nThe latter point regarding rules has been emphasized by ludologists in support of the concept of procedural rhetoric, which Bogost (2017) has touted as the core function of video games’ capacity to make meaning in a more substantial way than a gratuitous cut scene or superficial story to justify the strategy and action of gameplay (not unlike the largely irrelevant war signified in a chess match). However, our case studies will demonstrate that procedural properties and the game mechanic itself are vital to narrativity via the participatory properties. Despite the relative lack of agency in Bury Me, My Love, for example, players must advance the game themselves and must “interact” with the main character Nour—through small, sometimes quotidian, yet deeply humanizing simulated WhatsApp texts that advance the larger narrative—who is escaping Syria to seek asylum in Europe. This question of agency and freedom of player choice, or lack thereof, bears important implications for authorship to be discussed in each case. We contend that literacy is the key to gameplay in story-driven games, as our cases suggest the range of expression possible across various genres developed specifically to honor not only visual literacy, but the multiple forms of literacy (including spatial, kinetic, audio, and social media tools) in the twenty-first century that render narrative import through digital media.\n\nThis study therefore takes it as axiomatic that game mechanics, rules, and procedural properties, while often distinct from narrative content, can function as the engine driving interactive storytelling. The following case studies show how ludology is hardly distinct from narratology, but rather one and the same, inextricably bound in a mutually reinforcing relationship. The aim here is to complicate firm distinctions in formulations of games as constituting what Mayra (2008) describes a variety of “similarly interactive systems, with a specific emphasis on meaning-making through player action (ludosis), as contrasted with meaning-making as decoding of messages or media representations (semiosis), typical for such cultural systems as television show or contemporary poetry” (19). The diverse range of forms that video games can take indeed distinguishes them from television and contemporary poetry. Many of these forms exhibited in our four chosen games reveal ludosis, or how player action—mainly through text box choices uniquely contextualized and expressed through distinct graphic aesthetics and temporally adjusted interfaces—provides the kinetic agency of meaning-making as an act of decoding the media message (in this case narrative content). But the meaning is not in the tale, but in the telling; or as Marshall McLuhan said, “the medium is the message.” Gameplay for these and other story-driven games is tantamount to unraveling the narrative yarn. Studying narrative without examining the means by which they are put into play strips the game of its ludosis, just as examining game mechanics without reference to its story, particularly for narratively ambitious works such as Detroit: Become Human, denies its semiosis. As Herman and Vervaeck (2005) note, “the way in which a story is narrated … turns it into what it is.” He explains, “those who insist on denying the importance of the method of narration by reducing a story to content might just as well go to the movies or watch television because both of them can offer similar content” (7).\n\n\nMethods of analysis\n\nPurposive sampling led us to our information-rich case studies suitable for qualitative research in digital media such as this interpretive critical analysis of ludic narrativity in video games. It aims to achieve not total coverage or generalizability according to quantitative randomized sample studies, but “a deep, albeit partial and contingent, understanding of social reality in a specific context” (Lindlof and Taylor 2019, 143). Our sample is thus illustrative rather than definitive. In order to examine the leading edge of the genre, we selected four reality-based story-driven games that use ludic elements like text/dialogue boxes as the primary game mechanic that advances their narratives. The games were chosen to represent the various sectors of the global games industry at the leading edge of narrative video games: Detroit: Become Human by the French game developer David Cage and Quantic Dream (2017), Bury Me, My Love also by a French game developer Florent Maurin and Pixel Hunt (2017), Closed Hands by the British developer Dan Hett and PASSENGER (2021), and Chinese Parents by the Chinese developer Moyuwan Games (2018). These critically acclaimed games are all reality-based and employ distinct game mechanics to deliver complex narratives treating serious issues of learned debate with contemporary and historical significance. Detroit: Become Human is an allegory for U.S. slavery and the Civil Rights Movement (which anticipates the recent BLM protest movement); the Syrian refugee crisis is the focus of Bury Me, My Love; international terrorism constitutes the subject of Closed Hands; and Chinese Parents centers on Chinese collective memory of child-rearing from the perspective of parents.\n\nWe selected games featuring narratives bearing on social and political reality to indicate that a strong correlation exists between technical innovation in video game storytelling and the real-world relevance of their chosen topics. Although our case studies consist of titles that are technically designated as fiction, they are committed to social reality just as “a theatrical play may be documentary as easily as it is fiction,” as Aarseth notes (2016, 491). “For representational games,” he explains, “this means that as long as the game objects refer to events and existents in our world (e.g., in our history), they do not fictionalize but document.” The realistic import of these narratives heightens their urgency—as well as the precision of the affective and documentary nature of their telling—given their applicability to current political circumstances. We thus approach the fictional games of our case studies as bearing documentary functions with important sociopolitical implications, according to the formulation that “documentary simulations and games do not have to refer to specific objects to be documentary; they can also refer to generic objects (e.g., a type of airplane) and the resulting simulation is still documentary, not fictional” (Aarseth 2016, 491; Fullerton 2008).\n\nNarrative ambition unites our selected story-driven games, as reflected in the multiple endings possible in each, with six outcomes in Detroit: Become Human, 20 in Bury Me, My Love, nine in Closed Hands, and 100 in Chinese Parents. The rationale for our selection process prioritized games representing self-conscious attempts at achieving sophisticated narratives from across the global games industry, from mainstream AAA to indie and fine arts sectors. The various approaches to narratives in our sample thus showcase the adaptability of finely crafted narrative to different conventions and technological affordances. Originally released on PS4 and Microsoft Windows, Detroit: Become Human represents narrative innovation in the console game sector. The indie mobile game market’s foray into story-driven games appears in Bury Me, My Love, which is designed to be played on a smartphone, yet in an immersive way demanding extended engagement. As a conceptual game from the world of digital fine art, Closed Hands was designed for the PC specifically with use of headphones to enhance the game’s polished sound design carefully attuned to the nuances of the story. Whereas Bury Me, My Love and Closed Hands cater to niche audiences, Chinese Parents is a mass market production, which has won considerable market share in the highly competitive Chinese video game industry, particularly the casual indie RPG simulation sector. Thus diversity is represented in the sample in terms of aesthetics and gameplay as shaped by each title’s cultural, geographical, and industrial situatedness.\n\nThe multilinear and interactive nature of story-driven video games poses a challenge for narrative analysis (Plewe and Fursich 2018, 2473). Our method of critical analysis derives from approaches recommended by games scholars. With a focus on interface, rules, and goals, one approach examines entity manipulation (Zagal et al. 2005). A more holistic method considers not only formal and technical aspects, but also cultural and semiotic concerns (Konzack 2002). Our study’s focus on how technical aspects—figured according to ludology’s concept of procedural rhetoric—are inextricably bound to narrativity suggested the use of Carr’s (2009) method. Her approach draws from Barthes’s structural and textual method to recommend analyzing games as “playable text” at the levels of both rules and game mechanics as well as narrative content (Carr 2009, 1-2). We build on this method by proposing that rules and game mechanics perform narrative semiotic functions, offering a ludic grammar of interactive storytelling. That is, texts are not only playable according to Carr’s (2009) formulation, but from the reverse perspective, we posit that rules and game mechanics can be seen as textual insofar as they generate narrative through the player’s choices. Unlike Plewe and Fursich (2018), whose case study methodology separates analysis of rules and game mechanics from narrative content under the assumption that they are discrete entities, we combine treatment for each case. We posit that the game mechanic is the technological tool through which players access and engage the language of the game’s narrative, giving it shape through their own interactions.\n\nGames were examined for 1) synchronization of gameplay mechanic with narrative development, 2) formation of narrative structure, 3) thematic coherence, and 4) their impact on player experience. Additionally, paratextual promotional videos, playthroughs, reviews, and developer interviews and conference presentations were also accessed to contextualize the analytical process. Critical interpretation of formal aspects of the games is drawn from Aarseth (2003) and Zagal et al. (2005), particularly in terms of rules and game mechanics. Such technical features are vital to narrative tone and import, forming the empirical evidence to demonstrate the games’ ludic narrativity, defined as gameplay that tells a story through “units of meaning, or semes” (Carr 2009, 5). Player action, we argue, is the narrative glue that makes the disparate semes of the game cohere with what Carr (2009) calls “a binding quality,” one that forms “clusters and continuities with, across and beyond text” (5).\n\n\nThe (in)visible work of Detroit: Become Human\n\nDetroit: Become Human developer David Cage described writing the text for the game as a painstaking process of conceiving of its many different actions, consequences, and outcomes. Interactive writing, he noted, entails consideration of not only space and time, but also possibility. Each situation demands imagining thousands of variables, conditions, and options. Whereas film scripts typically contain roughly 100 pages, interactive stories like that of Detroit: Become Human can contain between 4-5,000 pages. Such sprawling narrative landscapes are essential to generating a plausible sense of player agency. In conscripting the player in the authorial role, gameplay becomes tantamount to an act of creative writing. Cage (2018) pointed out that the medium awaits the arrival of its own Stanley Kubrick or Orson Welles, because “Interactive storytelling can be what cinema was in the twentieth century—an art that deeply changes its time.” The goal for video games, as envisioned by Cage (2018), is to achieve the power and range of narrative expression previously associated with literature and film. Ian Bogost (2017) argued that such a goal is pointless for video games and anathema to their nature. In his view developers should instead focus on rendering subjects visible, operable, and perhaps even beautiful through gameplay. Detroit: Become Human thus defies Bogost’s (2017) assertions that “the future of games will be one in which games abandon the dream of becoming narrative,” and “to use games to tell stories is a fine goal, but it’s also an unambitious one.” The culprit, he claims, are walking simulators like What Remains of Edith Finch, a narrative masquerading as a game, which he believes would have been better told as an animated film.\n\nCage’s (2018) invocation of cinema as a standard of excellence defining the achievement of the medium’s maturity came shortly after—and arguably in response to—Bogost’s (2017) lament of “cinema envy” in the game industry. Story-driven games present a wrong-headed evolutionary trajectory of the medium, one that plays to another medium’s strengths while neglecting its own, particularly game mechanics and player choice, according to Bogost (2017). He decries this segment of “the game industry that has long dreamed of overtaking Hollywood to become ‘the medium of the twenty-first century,’ a concept now so retrograde that it could only satisfy an occupant of the twentieth.” Detroit: Become Human, however, uses the player’s choices to determine the outcome of six different endings. The game’s ostensible subject is its narrative, which gameplay indeed renders “visible, operable, and beautiful,” a process whereby storytelling itself on behalf of player choice is simultaneously ludic and semic (Bogost 2017). The game does not sacrifice action for contemplation. Choices are freighted with immense ethical pressure, which is intensified through the use of a time bar forcing the decision to be executed within seconds. The effect is to dramatize the difficulty of each decision, one that compounds and complicates the radical agency the game grants to players, who can precipitate war, peace, or go rogue. That intensity is compounded by the real-world implications of the plot.\n\nAs androids emerge into sentience, they become increasingly rebellious and strategic in their quest for freedom. Awareness of one’s own oppression as a necessary precondition to escape from slave labor echoes the pattern of Frederick Douglass’s liberation in Narrative of the Life of an American Slave. In it, Douglass details his enslaved childhood in the antebellum South where his captors systematically denied slaves literacy—and thus access to abolitionist writings—as a means of quelling rebellion. Once Douglass successfully teaches himself to read, he accesses abolitionist writings that enable him to comprehend the full extent of his oppression. This pattern appears in the game, which emphasizes the importance of sentience as the foundation of android empowerment. As with Douglass, androids’ awareness of the limits to their rights and freedoms fuels their escape and political activism. The gameplay dramatizes this gradually evolving awareness through a number of narrative choices that move the character through the storyworld by entering their actions on the controller. The desire to be free throughout the game is not abstract, but a response to a specific set of material conditions.\n\nSignificantly, player action occurs not as a question of self-indulgent personal preference, as with the gleeful anarchy and radical individualism of characters in Grand Theft Auto V, but with reference to the action’s impact on the larger android struggle for freedom. Gameplay in Detroit: Become Human therefore significantly departs from the narcissistic quest for self-aggrandizement and elevated social status driving games such as Fortnite, as seen in the quest for skins and character accoutrement that gratuitously capitalize on personal brand amplification endemic to digital culture and self-stylization (Dowling 2021, 94). Instead of leveraging player choice as an acquisitive form of consumer capitalism whereby skill is rewarded with faux capital via points that are visually valorized through badges, loot, and coveted skins, player choice in Detroit: Become Human is deliberately politicized. Whereas the path forward to liberation and equal rights appears in each player choice as a complex ethical dilemma—which presents a sophisticated understanding of how this and other social movements take shape through real decisions in isolated moments—the game neglects such careful consideration of why particular groups of humans have been (and continue to be) disqualified from the rights and freedoms enjoyed by others (Schubert 2021). As signaled in the title, the game’s forward-looking focus is on the challenge of becoming human, providing a meditation on the influence of individual choice on racial justice in the U.S. Nothing less than the fate of an oppressed minority rides on player choices, each of which are situational, temporally limited, and chart a unique narrative course.\n\nAgency is central to Detroit: Become Human’s gameplay mechanic. Whereas players must have quick reactions in physical conflicts and for complex tasks, their more ethically laden decisions that directly shape the narrative occur through text box options that allow the player to choose what to say or do. As Schubert (2021) observes, “narrative and gameplay in Detroit: Become Human are tightly interwoven and hard to separate.” The game’s mechanics thus catalyze “a larger, interconnected system by which a digital story takes shape” (Green 2017, 12). For example, the gathering of evidence for “The Hostage” chapter that opens the game conscripts the player in the role of co-author of the narrative. The nature of the evidence gathered by the player dictates the outcome of the chapter, precisely because that data determines Connor’s approach to rescuing a hostage taken by a deviant android. The game mechanic does not allow the player to overpower the deviant android, but instead requires the player to interpret the evidence available in the storyworld to achieve a safe resolution. As Schubert (2021) notes, “gameplay decisions in Detroit: Become Human are (almost always also) narrative decisions” (12). Quicktime events and dialogue choices extend the narrative branches. The branching story co-authored through gameplay is displayed at the end of each chapter. This detail underscores the importance of narrative as the main accomplishment or product of gameplay, which is constructed as a semantic exercise in critical thinking and interpretation of the simulated environment.\n\nThe individualism of a traditional first-person shooter, as in battle royale games like Fortnite, ends in the death of the player’s avatar. By contrast, the death of the player’s avatar in Detroit: Become Human does not end the game, as play may continue to support the plight of androids through the two other playable characters. Narrative construction is thus showcased as the core value of gameplay, as Cage and his designers preserve the player-generated story by continuing, rather than erasing, progress upon a protagonist’s death. The ludic narrativity of android liberation can thus be constructed through multiple protagonists.\n\nPlayer investment in shaping the through line of the unfolding story of Detroit: Become Human is reinforced through flowcharts which appear at the end of each chapter. The flowcharts allow characters to rewind certain scenes to either recant decisions or lament them. This detail positions gameplay as a form of authorship, not in the abstract, but in the literal telling of the fate of the liberation movement. Conner’s character is not locked into his programming to suppress the liberation; he may deviate from it to aid the uprising, but can also abruptly decide to shoot Markus at his speech during the final android protest. Many chapters contain six different endings. The flowcharts following each chapter highlight not only the decisions players made, but also those they did not. The otherwise invisible work of interactive writing from the designer’s perspective becomes entirely visible to the player, who is invited into this dimension of the process of game production. In the process of gaining new awareness of interactive narrative production from the scriptwriter’s perspective, player consumption is enriched through a new self-awareness of their own choice pattern whereby they might elect to pursue different paths on the next playthrough. The narrative blueprint’s appearance at these key intervals echoes the flowcharts frequently used by novelists and narrative nonfiction writers. A plot chart similarly plays a prominent role in Closed Hands, a game in which the player becomes acutely aware of unchosen paths and their alternative consequences. Detroit: Become Human thus epitomizes the principle of gameplay that entails revealing consequences of actions to players within the context of the storyworld. As Murray (1997) explains, agency is “satisfying” when we “see the results of our own decisions and choices” (126).\n\n\nThe performative narratives of Bury, Me My Love\n\nThe parasocial “trying on” of alternative identities can raise player awareness and empathy for disadvantaged and systematically oppressed populations. As shown in Detroit: Become Human, identification with marginalized game characters can become what Leach and Dehnert (2020) call “a means of exploring the social locations and experiences of marginalized others,” especially in the construction of racial identity and strong female protagonists (23). Bury Me, My Love (which translates from take care; you better not predecease me in Arabic) similarly immerses players into the experience of a marginalized population, particularly through the reality-based journey of a Syrian refugee migrant. Rather than recreating a visually ornate and elaborate storyworld to simulate that experience, Florent Maurin and his team of developers of Pixel Hunt designed the game from the perspective of the migrant’s loved one, as told through this digital correspondence in a recreated version of a WhatsApp interface. Players are recommended to experience the game on their phones to better approximate the communication between the migrant woman Nour and her husband Majd at home. That correspondence takes on a wide range of subjects and tones, from playful and intimate to serious and strategic depending on Nour’s situation along her journey. The result is a pattern of gameplay that advances the narrative via the communication with Nour, who faces desperate and life-threatening circumstances that the player must guide her through from the perspective of Majd.\n\nThus traditional gameplay is undermined on several fronts in Bury Me, My Love, each of which contributes to a unique form of ludic narrativity. Majd in this game can be construed as the player’s avatar, as the player guides Nour to safety through him and all gameplay choices dictate the content of his communication. The effects of player choices, according to Murray’s principle of best practice for narrative-driven games mentioned earlier, are immediately apparent in how they shape Nour’s condition. Majd—and thus the player—is tasked with a challenge fraught with violence and mortal risk that the developers conscientiously elected to portray off scene, or at least metonymically through the WhatsApp exchanges that take place through text box options. In addition to player-avatar relations to enhance the parasocial functions of the narrative, Bury Me, My Love’s adopted medium of the mobile games is not typically associated with immersive, story-driven titles. A deliberately non-visual interface for scene construction paradoxically heightens the emotional intensity of communication choices that constitute the gameplay mechanic.\n\nThe reality-based ludic narrativity of Bury Me, My Love was originally inspired by Lucie Soullier’s Le Monde article, “The Journey of a Syrian Migrant as Told by her WhatsApp Messages.” An experience told through a record of social media correspondence is impressive in print, but takes on an entirely new feel when such messages are activated on one’s own phone in the role of Majd. The character and player thus become one through the correspondence with Nour. As developer Maurin (2021) noted, “using interfaces people are already used to is powerful,” particularly in terms of its temporality, which is manipulated “to create a close bond between characters.” This form of gameplay that places the player in such close proximity to events crafted to replicate those of the real immigrant experience carries the potential of becoming emotionally overwhelming. Given the gravity of the content in Soullier’s nonfiction report, for example, the team of developers at Pixel Hunt chose not to include the topic of rape in the game, but elected to include Nour’s death in several of the game’s twenty different endings. According to Maurin (2021), drawing many of the twenty narrative threads to the passing of Nour was a decision made out of respect, which is depicted through audio clips that mark the end of the playthrough. “She’s fictional,” he explained, “but represents the real lives that are being lost” (Maurin 2021). Such serious content is uncommon—if not unprecedented—in the mobile games industry, which leans instead toward more casual content designed for diversion. In this case, the mobile game highlights the use of the smartphone in the migrant experience as the lifeline to many refugees’ relationships and a key tool enabling their passage to safety.\n\nAlthough Nour’s survival depends on the narrative path assigned to the player, no combination of choices made by the player has a bearing on the outcome. The gameplay leading to twenty possible endings of Bury Me, My Love is therefore pre-scripted. “The player has no agency,” according to Maurin (2021). “Everything is decided by hidden variables,” he explained, unapologetically adding that “some players complain that they don’t have enough agency.” Indeed, most of the choices in the text boxes may shape the tone of the correspondence with Nour in any given scene, but they in all cases do not determine the outcome. Maurin’s rationale for this design is premised on the notion that story-driven games can be performative rather than choice driven. The real-world significance of the topic led Maurin to strip agency from the game, because the question of winning is irrelevant in his view. The forced conclusions metaphorically convey the lack of agency faced by migrants. “Some immigrants do succeed and others don’t,” he urged, noting that the game raises the questions, “whose stories get told? Who is allowed to tell these stories?” (Maurin 2021). Narrative thus appears as performative and pre-scripted in order to focus gameplay on the immediate characters as a window into the larger Syrian refugee crisis. This lack of agency should not be viewed as a limitation or liability, but rather one of the game’s many innovations described above, which include: 1) a powerful female Arab protagonist, 2) immersive, story-driven, serious, reality-based content for a mobile game, 3) violent content (via text and audio) that is not visually depicted 3) a simulated social media interface for the gameplay mechanic, and 4) a narrative structure consisting of twenty separate vectors (with brief side branches) that progress chronologically.\n\n\nClosed Hands\n\nBury Me, My Love shares with Dan Hett’s Closed Hands the overarching goal of raising player consciousness about the personal impact of political violence. Unlike Bury Me, My Love’s mobile design and performative narrativity with limited agency, Closed Hands offers radical player agency in shaping the selected protagonist’s narrative. Character fates do not play out on separate vectors, but can be intertwined based on player choice, predominantly through text and dialog boxes. As with Detroit, Become Human, player agency is positioned as an interpretive act demanding thoughtful and conscientious literacy from the player. Developer Dan Hett, who lost his brother in the 2017 Manchester terrorist bombing, structured the game around the event, which is not shown visually. Players choose from one of five characters with the option of switching among them at any time, as the narrative flowchart is accessible at any moment during gameplay. Too large to fit on the screen at once, this story tree of the game’s intricate plot options can be explored by clicking and dragging to unseen portions of it. As with the flowchart feature of Detroit: Become Human, this overview of the completed nodes in the plot sequence signals the effects and consequences of gameplay to the player. At the center of the flowchart is a box labeled “Incident,” a fictional terrorist bombing in a British city from which the player can select a node in the network featuring the experience of one of the characters (Hett 2021). Gameplay incentivizes completion of each node which opens up a new, previously greyed-out, branch of the story tree. This game mechanic is figured as an act of unveiling a politically significant plot that intertwines with the other characters. One must play and complete each node to open a new thread with the goal of reaching the end of the story, upon which the player sees the message: “You’ve reached the end of a story—one of them. Closed Hands is an intricate story with many threads. Although you’ve reached the end of this one, others remain” (Hett 2021). Gameplay thus proceeds with a distinctly novelistic feel whereby immersion is narrative rather than visual, as the technology never aims for graphic or audio simulation of any event.\n\nThe sense of being captivated by each of the storylines of Closed Hands is thus driven by the profound personal intimacy rendered in each character portrait. Sound design accentuates narrative intrigue aiding shifts in tone during any given sequence without being overbearing or melodramatic. Intimacy is achieved through scene and dialogue depicting personal, professional, and civic aspects of each character through simulations of their digital lives via instant messaging chats with co-workers and loved ones as well as menacing exchanges with threatening figures. As an alternative to featuring embodied avatars that the player moves throughout a storyworld, key aspects of characters are depicted visually through their faux digital desktops. At various stages in gameplay, the player occupies the digital desktop of each of the five main characters. Mike, a middle-aged British white male with racist inclinations associated with the Patriots group who threaten the Muslim community in the wake of the bombings, has a desktop background image featuring a giant football (soccer) stadium; Farah’s desktop background bears an official government seal reflecting her work for the Police Intelligence Service. Simulated digital interfaces provide the dramatic stage and gameplay interface for characters’ online communication. Haziq, the Muslim shop owner and father of a perpetrator of the bombing, at one point accesses his son’s computer and communicates via instant messaging with a friend of his son to ascertain his whereabouts after he had gone missing; Beth, a reporter at odds with her editor who pressures her to sensationalize the story, voice texts with him from the scene of the bombing; Markus Bashir, an eyewitness of the event who advocates for a compassionate response to the Muslim community, receives a threatening message from the Patriots that escalates into a heated correspondence on his laptop (Hett 2021). By simulating their digital behaviors via the use of their online tools, these interfaces immerse the player deeper into the characters’ personal lives.\n\nThe greatest degree of player agency in Closed Hands is experienced in opportunities to select the next scene within the space of the flowchart. The choices within each scene—by contrast to the radical autonomy offered when selecting scenes and charting a unique narrative web—are essentially unilinear, locking the user into a fixed outcome. Choices in the dialogue box render the same outcome but are designed to appear different, a feature that is often thinly veiled by synonymous options like “transparency” and “honesty” in one case, and “I think so” and “I hope so” in another (Hett 2021). Such buttons are like scrolling down in a multimedia narrative to reveal the next screen, which is essentially a form of digital page-turning. Yet scene selection on the flowchart offers opportunities to move forward or backward through time and to switch to any of the other four protagonists. The larger sweep of the narrative constructed by the player is not lost upon completion. The coda of the endings in Closed Hands as played from Haziq’s and Markus’s perspectives features a reflection back on the event with the reporter Beth, a detail that places the significance of the attack into perspective from the vantage point of ten years in the past for Markus.\n\nThe gameplay of Closed Hands generates authentic player interest and curiosity in the various perspectives and interrelated stories of these characters, one bearing heavily on the precarious position of the Muslim community in a British town following such a tragedy. Among the most poignant moments reinforcing them are Haziq’s attempt to reconcile with his son’s crime. Through Haziq, players experience the tragedy as a member of the Muslim community, which is then mediated to the general public through Beth’s reporting. Haziq identifies with Beth’s portrayal of his son Yasid as a “confused, weak boy,” a moment that brilliantly harkens back to her journalistic past as a principled idealist who defied her editors. The mainstream press’ pursuit of profit and audience supersedes concern for community voice, as she discovered early in her career when she covered political protests. Her editors thus implored her to “run it big language on the topper—bang up the drama.” In response to her insistence to wait until casualty figures could be confirmed, the editor asks “Do you wanna be the first to the story or the best but last?” (Hett 2021). This final point suggests the keynote and overall objective of Closed Hands as a media product designed to defy the manic, Twitter-driven news cycle’s superficial and amplified reportage. This self-reflexive moment alluded to the game’s own slower, more nuanced process of production of an alternative interactive form of immersion into the layered, complex, and interconnected meanings of a terrorist bombing, wherein the personal bleeds into the political.\n\n\nChinese Parents and storytelling of life course\n\nOur last case is Chinese Parents, a simulation game that allows players to experience the journey from infancy towards adulthood as a Chinese child. Players have 48 turns to shape the life of their role-playing character. Their choices will affect the character’s statistics (e.g., IQ, EQ, memory, and stress level); statistics will affect the cost of skills learning, and the final statistic and skills acquired will determine what kind of life the role can have at the end. Choices need to be made in each round on how to plan the character’s schedule. Different arrangements impact the character’s statistics in different ways. For example, pushing your character to excel in academic fields will increase IQ. However, it will also add up the stress level. When under excessive stress, the character may experience serious mental issues which will result in game over. Playing Nintendo games can help relieve stress, but it will lower parents’ satisfaction. Since every choice has a trade-off, there is no easy way of gameplay, just like there is no easy mode of life, especially for new players. In addition to the player's scheduled events, there will be unexpected incidents every round, such as “being the last kid to be picked up after school.” These incidents can also affect the role’s statistics in a certain way, thus adding more unpredictability to the character’s life. After the first playthrough, the player can start a new cycle by role-playing the next generation.\n\nIn general, the game accurately depicts the life course and captures many shared experiences of Chinese children, such as working hard to meet the high expectations of their “tiger mother,” preparing hard for the ultimate challenge: Gaokao (the university entrance exam). It especially reflects the childhood of those who were born between 1980-2000. Growing up in a time that had seen an unprecedented fast economic expansion and social upheaval in Chinese history, this generation had no memories of living in abject poverty, and collectively received a good education and had relatively unrestricted access to information. Considering this, it is no surprise to see the character in the game never experience any pain of hunger or poverty. Another interesting detail is that the character has no siblings. This goes in line with the experience of those post 80s and 90s, since they are the first generation affected by the one-child policy, which limited most Chinese couples to one offspring each (Lian 2014).\n\nIn this sense, the story told by this game is actually the story of a person’s life, or more precisely, the collective memory of a generation. By allowing players to step into the shoe of a typical Chinese child and play both the role of parents and the kid, the game gives the grown-up post 80s and 90s a chance to rethink their lives and their relationship with their parents.\n\nThis narrative goal is highly supported by the game mechanics of Chinese Parents, whose gameplay is heavily based on turn-based strategy. Each turn, players select a course of action from a text box that displays the available strategy options, click to execute, and the system automatically completes all actions for that turn and gives feedback. Much of the feedback is presented in the form of event summary in a pop-up text box, e.g., “Dad gets home drunk and starts shouting a large variety of well-selected curse words at you.” This event causes the character's intelligence attribute to be reduced by five. With this game mechanic, Chinese Parents organically integrates a large number of events that pass collective memories into the player's game experience.\n\nThis mechanism effectively increases the narrative speed, making it possible to tell the story of a growth spurt of about twenty years in about three hours. It also means that the game can have a higher narrative density, as more anecdotes can be incorporated into the story. More crucially, by adopting this mechanism, the telling of events in this game is freed from the constraints of traditional storytelling structures, which usually require the chosen events to form a high-level story structure such as a three-act structure. This is a challenge for role play games, especially for sandbox games whose plot is highly determined by players. The player may spend ten minutes doing nothing but wandering, resulting in a personal story which is far from interesting. In Chinese parents, the events are also not organized into a drama structure. If the game were transformed into a text, the result would be a daily stream of events with flat characters and a monotonous plot, rather than an interesting, coherent story. However, once played, it’s totally different. What a simple narrative like “Dad gets home drunk and starts shouting a large variety of well-selected curse words at you” presents is not just a daily incident but one that could result in a reduction of the character’s intelligence. The subsequent occurrence may have nothing to do with the preceding incident in reality, but it may exacerbate or mitigate the intellectual harm caused by the preceding event in the game. Events that lack connection in reality can constitute a meaningful and coherent experience for the character in the game.\n\nThis highlights a distinctive storytelling capability of video games: they are able to communicate stories that are challenging to convey through conventional narrative techniques. Many human experiences are frequently ignored because they are difficult to logically incorporate into conventional plot structures or dispersed across numerous characters. Games, with the aid of their own particular rules, can save these human experiences and bring them back to light. Therefore, when playing the game, players are not just retracing their steps but rather experiencing it in a new setting with a fresh perspective, seeing and remembering many of the things they had previously overlooked. This could prompt players to reevaluate important social issues like the relationship between parents and children, what constitutes a good education, what constitutes a successful life, etc.\n\n\nConclusion\n\nThis study revisits well-trodden ground with a new goal in mind: to situate reading a game in relation to ludology vs. narratology debates. Reading as a mechanic helps reveal how ludology and narratology are mutually reinforcing rather than mutually exclusive categories in each of the ensuing case studies. The first of the cases considered is Detroit: Become Human, which represents a visually robust, action-based blockbuster noted for its narrative prowess by industry critics and media scholars (Holl & Melzer 2021; Leach & Dehnert 2021; Schubert 2021). On the surface, the game bears the appearance of a dystopian action video game, yet its gameplay mechanics are concerned more with conscripting player agency in building narrative through ethical choices than kinetic combat as in a first-person shooter. Three text-based games comprise the next case studies, Bury Me, My Love; Closed Hands; and Chinese Parents, illustrating how procedural rhetoric, defined as meaning-making through player action, constitutes the engine driving each game’s narrative. These cases present strong evidence suggesting that the leading edge of story-driven games converges narrativity with player choice rather than treating them as discrete entities. Findings counter the ludologists’ charge that storytelling is anathema to video games, a view that casts narrative’s place in the medium as a facile, gratuitous window dressing superimposed on the game mechanic. The ludologists’ core premise, that the medium of video games essentially generates meaning through exploratory player action shaped by rules, can apply to the co-construction of narratives.",
"appendix": "Data availability\n\nNo data are associated with this article.\n\n\nReferences\n\nAarseth E: Genre Trouble: Narrativism and the Art of Simulation.Wardrip-Fruin N, Harrigan P, editors. First Person: New Media as Story, Performance, and Game. Cambridge, London:The MIT Press;2004; pp. 45–55.\n\nAarseth E: Ontology.Wolf M, Perron B, editors. The Routledge Companion to Video Game Studies. New York:Routledge;2016; 484–492.\n\nAarseth E: Playing research: Methodological approaches to game analysis. Paper presented at Melbourne DAC: 5th International Digital Arts and Culture Conference, Melbourne, Australia, May 19-23. 2003.\n\nBogost I: Video games are better without storytelling. The Atlantic. 2017 10 April.\n\nCage D: How video games turn players into storytellers [video]. TED Conferences.2018.Reference Source\n\nCarr D: Textual analysis, digital games, zombies. Paper presented at DiGRA 2009 Conference: Breaking New Ground: Innovation in Games, Play, Practice and Theory, West London, UK, September 1-4. Chinese Parents [video game]. Shanghai: Coconut Island Games. 2009.\n\nDowling D: The Gamification of Digital Journalism: Innovation in Journalistic Storytelling. New York:Routledge;2021.\n\nEgenfeldt-Nielsen S, et al.: Understanding Video Games: The Essential Introduction. New York:Routledge;2020.\n\nFrasca G: Ludologists love stories too: Notes from a debate that never took place. Proceedings of DIGRA. 2003; pp. 92–99.\n\nFrasca G: What is ludology? A provisory definition. Ludology.org. 2001.\n\nFullerton T: Documentary games: Putting the player in the path of history.Whalen Z, Taylor L, editors. Playing with the Past; History and Nostalgia in Videogames. Nashville:Vanderbilt University Press;2008; pp. 215–238.\n\nGreen AM: Storytelling in Video Games: The Art of Digital Narrative. Jefferson, N.C.:McFarland;2017.\n\nGrodal T: Stories for Eye, Ear, and Muscles: Video Games, Media, and Embodied Experiences.Wolf M, Perron B, editors. The Video Game Theory Reader. Routledge;2003; pp. 129–155.\n\nHerman D, Vervaeck B: Handbook of Narrative Analysis. Columbus:Ohio State University Press;2005.\n\nHett D: Closed Hands [video game]. Manchester:PASSENGER;2021.\n\nHoll E, Melzer A: Moral minds in gaming: A quantitative case study of moral decisions. Detroit: Become Human. Journal of Media Psychology: Theory, Methods, and Applications. 2021; 1–12.\n\nJamison L: The enduring allure of choose your own adventure books. The New Yorker. 2022 12 September.\n\nJuul J: Games telling stories. Game studies. 2001; 1(1): 45.\n\nKonzack L: Computer game criticism: A method for computer game analysis.Mayra F, editor. Computer Games and Digital Cultures Conference Proceedings. Tampere:Tampere University Press;2002; pp. 89–100.\n\nLian H: The post-1980s generation in China: Exploring its theoretical underpinning. J. Youth Stud. 2014; 17(7): 965–981.\n\nLindlof TR, Taylor BC: Qualitative Communication Research Methods. Thousand Oaks, CA:Sage;2019.\n\nLeach R, Dehnert M: Becoming the other: Examining race, gender, and sexuality in Detroit: Become Human. Rev. Commun. 2021; 21(1): 23–32.\n\nMaurin F: Panel discussion: Complex storytelling in games. Push Festival 2021, 10 March. Manchester UK Council for Arts.2021.Reference Source\n\nMayra F: An Introduction to Game Studies. Thousand Oaks, CA:Sage;2008.\n\nMurray J: Hamlet on the Holodeck: The Future of Narrative on Cyberspace. Boston:MIT Press;1997.\n\nNelson X: The evolving art of dialogue in video games. 16 April PC Gamer. 2018.Reference Source\n\nPlewe C, Fursich E: Are newsgames better journalism? Empathy, information, and representation in games on refugees and migrants. J. Stud. 2018; 19(16): 2470–2487.\n\nRyan ML: Narrative across Media: The Languages of Storytelling. University of Nebraska Press;2004.\n\nRyan ML: Narrative as Virtual Reality: Immersion and Interactivity in Literature and Electronic Media. Baltimore:Johns Hopkins University Press;2001.\n\nSchubert S: “Liberty for Androids!”: Player choice, politics, and populism in Detroit: Become Human. Eur. J. Am. Stud. 2021; 16(3): 1–24. Publisher Full Text\n\nTompkins A: Acts of becoming: An examination of historical symbolism and embodied empathy in Detroit: Become Human. Loading. 2021; 14(24): 1–25.\n\nZagal JP, et al.: Towards an ontological language for game analysis. Paper presented at DiGRA 2005 Conference: Changing Views—Worlds in Play, Vancouver, Canada, June 16-19. 2005."
}
|
[
{
"id": "159900",
"date": "06 Feb 2023",
"name": "Dominic Arsenault",
"expertise": [
"Reviewer Expertise Game studies",
"narratology",
"interactive narrative",
"history of video games",
"aesthetics",
"film studies",
"animation film"
],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe paper provides an analysis of 4 games that were identified by the authors as providing compelling cases that the opposition between ludology and narratology is thin, and perhaps an unproductive way to view video games. The first part of the paper describes the opposing views from proponents of a narrative understanding or approach to video games, and those favoring game mechanics and other terminology or approaches that place the focus of their investigation on the interactivity of games. The main argument is sensible and well-presented.\nThe viewpoints, arguments and stances from each camp are adequately summarized. There may be a bit of caricaturing or simplifying of views, which is understandable as much has been written on the subject, but still could be worded a bit better at times. This is a minor perfectible area of the paper.\nTo game studies specialists, the time spent in exposing the two conflicting positions of narratology and ludology will probably look like an overly long re-treading of an all-too familiar debate, as the question has been addressed for more than twenty years now. I recommend the authors make it more explicit in the introduction that the part “theorizing ludic narrativity” presents the debate, more particularly to the benefit of readers who may not be aware of the historical development of game studies during the 2000s. I also recommend the author(s) amend the abstract, which claims that “the narrative potential of video games is still being debated”. The debate seems moot, and the most recent writing the author(s) share to that effect is an almost 6-year old text by Bogost in a newspaper.\nThese minor issues aside, there is a more substantial hurdle in the paper’s general logic and contribution. Overall, the paper’s argumentation seems like a self-fulfilling prophecy; the time spent in setting up the conflicting positions of narratology and ludology is not well spent since the author(s) have chosen four games that do indeed focus on storytelling rather than mechanics. I appreciate the authors' claim that reading and following the narrative is itself a ludic act / toolkit and vice-versa, but I think the framing context to get to that claim is on an off-axis base. Rewording the first pages would help to streamline the overall thrust of the paper.\nThe ”Theorizing ludic narrativity” functions well as a primer for readers unaware of the field of game studies, and the next argumentative move is pertinent in illustrating the ways in which modern games can tackle the issues of narrative progression and narrative comprehension intermingled with the interactive experience.\nThe analyses of the four games is a great contribution this paper proposes. The writing is sharp and focused, the points are well-made and the work is illuminating. Overall, the fourth analysis (“Chinese Parents and storytelling of life course”) would benefit from some rewriting and a bit of expansion, if the space allows. It offers less substance than the other three analyses and the writing falls a bit short. The section is descriptively rich (though the familiar language, as in “Once played, it’s totally different.”, contrasts with the previous ones), but I was expecting a more substantial discussion of the implications of these choices and particularities for storytelling with regard to game storytelling. What’s there is good, I was simply expecting a bit more.\nOverall, the paper offers a good contribution through its analyses. It also provides a service to non-game studies scholars for its quick and readable survey of the issues in games and storytelling. Some minor rewrites would be beneficial. A minor rewrite and expansion upon a part is needed for publication. I recommend indexing after these minor revisions are done.\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Partly\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nNot applicable\n\nAre all the source data underlying the results available to ensure full reproducibility? No source data required\n\nAre the conclusions drawn adequately supported by the results? Yes",
"responses": []
},
{
"id": "162890",
"date": "01 Mar 2023",
"name": "Edmond Chang",
"expertise": [
"Reviewer Expertise Video game studies",
"analog game studies",
"queer game studies",
"literature",
"pedagogy",
"\"close playing",
"\" digital humanities",
"popular culture"
],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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, Chen, Dowling, and Goetz's research article attempts to further resolve the now decades long theoretical feud between narratology and ludology. The article argues that the two sides are in fact complementary, even supplementary through what they name as \"ludic narrativity,\" a useful analytic that narrative is a game mechanic. I very much appreciate the survey of games--Detroit: Become Human, Bury, Me My Love, Closed Hands, and Chinese Parents--because they offer a range of genres, platforms, topics, and represent global developers.\nMuch of the article is devoted to the \"close playing\" of Detroit: Become Human. I pause at the conflation of android and slavery and at the invocation of Frederick Douglass without a more careful contextualization of race, gender, and identity in the game. This reader would love to see further contextualization of race, gender, identities, and inclusion of more close playing details across all of the games.\nI do appreciate the \"readings\" of Bury, Me My Love, Closed Hands, and Chinese Parents, particularly when the mechanic is to limit player choice, to require the narrative to be directive. More on how these tensions work across and between the games would provide a firmer account of the \"ludosis\" of the texts. The unpacking of the \"ludic grammar of interactive storytelling\" in D:BH and other games is compelling, though I am ambivalent about (perhaps agnostic regarding) the linking even eliding of play, choice, player agency, and literacy.\n\nThis respondent questions the immersive fallacy (a la Salen and Zimmerman) and by extension what I have called the interactive fallacy wherein player choice, player agency, the ability to navigate interface, decision trees, and discover different endings belies the permissions, prescriptions, and protocol (cf. Galloway) always-already predetermined by the game's code. That said, I would like to know more about the \"conscientious literacy\" raised by the article and how that intersects or updates the arguments made by edu-game scholars like James Paul Gee or how that differs from the slide toward gamification: is reading and playing the same thing? Is playing and \"authorship\" homologous1? The notion of the \"text box\" as mechanic and readerly/playerful interface is really interesting and could be foregrounded more. Moreover, given the focus on how text and story is deployed by each game, we do not see much close reading of the language and narratives themselves.\n\nFinally, I appreciate the response to Bogost's polemic and wonder if that contention is too binary, too. I do think the article could incorporate and respond to some more recent work by queer game scholars and scholars of color. As with the concern over immersion, the desire to read and play games as \"empathy machines\" could be interrogated further via the work by Kishonna Gray, Anna Anthropy, Tanya Pobuda, Bo Ruberg, and others.\n\nOverall, the article is thoughtfully written, accessible, and the case studies will be of great use to game scholars, developers, and players.\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? No source data required\n\nAre the conclusions drawn adequately supported by the results? Yes",
"responses": []
},
{
"id": "162888",
"date": "14 Mar 2023",
"name": "Christopher Paul",
"expertise": [
"Reviewer Expertise Game studies",
"rhetoric"
],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe central argument of the essay is that narrative and game mechanics, or narratology and ludology, actually are better considered together rather than separately.\nThe essay uses four games as core examples, including titles like Detroit: Beyond Human and Chinese Parents. Also rooted in the history of game-like aspects of storytelling as found in Choose Your Own Adventure books and narratives with multiple endings like the “Bandersnatch” episode of Black Mirror, the essay pushes against the notion that games cannot tell compelling stories.\nThe authors make the case that, in video games, the game mechanics are actually a key part of storytelling than inform and alter the narrative dynamics and expand the potential of games. I agree!\nMiddling questions like this and pursuing a both/and option, rather than an either/or approach is generally more appealing to me than trying to force a dichotomy and place a required choice on players and critics.\nI would also push the essay in two primary ways.\nFirst, I’m a big fan of the idea that games are more than just stories or game mechanics. I typically push to studying the way that words, game design, and play all work together to make meaning in games. That makes this essay interesting to me, but I also think there’s a dynamic of play and how the player interacts with the game that is not fully accounted for in this piece.\nTo that end, in the repeated example used in discussion of Chinese Parent, the dad coming home drunk and swearing at the player is a short text prompt that changes the game mechanics but can also be made more meaningful by the interaction of that prompt and the player’s experience. It’s not just mechanics. It’s not just story. It’s the interaction of those in combination with play that help signify how meaning works in games.\nAs an example, there are stories told by players in and around sports games where players develop their own narrative and story around the game in a rich interaction of imagination, storytelling, game mechanics and play. Ricky O’Donnell’s chronicling of his version of the Western Illinois basketball team or the Football Manager players who ply their trade in fictional countries like Morgsthia push on narratology, ludology and the middle approach of claiming both matter by underscoring the role of play and playfulness in understanding how games are made meaningful.\nSecond, although I think there is plenty of room to critique Bogost’s approach and the role of procedural rhetoric in game studies, I also think this paper sometimes 'straw person’s' that argument a bit. This kind of critique of game narratives is often not that games cannot tell stories, but that they don’t tell *good* stories. With that in mind, it is hard to make the case that “Bandersnatch” is the best episode of Black Mirror or that Choose Your Own Adventure books are paragons of literary excellence. Instead, they are novel explorations of a different mode of storytelling that frequently get left in the dust by more typical approaches to narrative design, whether that be in episodes like “San Junipero” or young adult novels like The Westing Game.\nA primary contribution of this work is in the discussion of the case studies. The breakdown of the games is really interesting and advances my understanding of each of them. Beyond that, I love how the selected games are not necessarily ones I have already read a ton about. Pulling a diverse array of interesting games and elevating them is worthy of discussion and exploration. So, let’s move past narratology and ludology and consider games on their merits. What is interesting about them? What can we learn from them? And how can we better understand them and how they make meaning?\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? Partly",
"responses": []
}
] | 1
|
https://f1000research.com/articles/12-45
|
https://f1000research.com/articles/12-44/v1
|
11 Jan 23
|
{
"type": "Research Article",
"title": "Equatorial Plasma Bubbles Identification with Single Frequency GNSS Receiver Data",
"authors": [
"Tatiparti Padma",
"Swapna Raghunath",
"Usha Kumari Chintalapati",
"Swapna Raghunath",
"Usha Kumari Chintalapati"
],
"abstract": "Ionosphere is the highest contributor of positional error in Global Navigation Satellite System (GNSS) owing to the trans-ionospheric signal delay, distortion and possible outage. Equatorial and Low latitudinal ionosphere experiences frequent perturbations in plasma density most of which is due to the presence of Equatorial Plasma Bubbles (EPBs). In order to improve positional and navigational precision of GNSS signals, EPBs must be identified and corrected. EPBs begin to manifest post-sunset and continue to thrive past midnight. This paper proposes a Rate of TEC Index (ROTI) based EPB identifier using single frequency GNSS receiver data. GNSS data has been collected from the archives of Scripps Orbit and Permanent Array Center (SOPAC). The raw data has been downloaded from the global continuous network station, IISC, located at Bangalore (13.0219°N, 77.5671°E) in Receiver Independent Exchange (RINEX) format. Analysis of EPB occurrence has been carried out for six consecutive years in the second half of the 24th solar cycle (2014-2019). The results show a clear surge in the number of EPBs identified in the course of equinoxes.",
"keywords": [
"GNSS",
"Equatorial Plasma Bubble",
"International GPS Service",
"Rate of TEC Index",
"Total Electron Content"
],
"content": "Introduction\n\nGlobal Navigation Satellite System (GNSS) is a satellite system that is used to pinpoint the location of a user’s receiver anywhere in the world. International GPS Service (IGS) is a worldwide network of receivers tracking the transmissions of GNSS satellites is a valuable source of ionospheric data that is globally distributed and continuously available.1 The services provided by GNSS are used in massive number of applications, both civilian and military. However, these signals are prone to several sources of disturbances, causing errors which result in degradation of positioning accuracy.\n\nIonosphere is the layer of the earth’s atmosphere extending from 80 to 1,000 km above the earth’s surface containing a high concentration of ions and free electrons and is able to reflect radio waves. Free electrons and ions are produced by solar radiation which causes group delay and phase advancement of GNSS signals resulting in positioning errors in order of tens of meters. Ionosphere is the highest contributor of GNSS positioning error.2 The ionospheric F-region extending from 250 to 400 kms contains the greatest concentration of free electrons. The plasma disturbances in the F-region scatter radio waves and generate rapid fluctuations in the amplitude and phase of GNSS signals called scintillations. The disturbances in ionospheric plasma are most frequent and severe in the geomagnetic equatorial belt from 20°N to 20°S, particularly during the post-sunset hours. These scintillations depend on the ionospheric Total Electron Content (TEC) which is the total number of free electrons in a cylindrical volume of one square meter cross section, extending from the receiver to the satellite.3\n\nLow-latitude ionospheric scintillations are season- dependent and are primarily limited to local night-time hours.4 The ionospheric plasma irregularities vary greatly in spatial extent and drift with the background plasma at speeds of 50 to 150 m/sec. Ionospheric effects on GNSS signals are complex and difficult to mitigate given the inhomogeneous and anisotropic nature of the medium.5\n\nEquatorial Plasma Bubbles (EPBs) represent plasma depletions with respect to the background ionosphere and are the most important sources for electron density irregularities in the F-region.6 EPBs are the ionospheric phenomenon near the earth’s geomagnetic equator which makeup one of the distinguishing phenomena that disrupt radio and satellite communications. EPBs are generated through the nonlinear evolution of the Rayleigh-Taylor instability (RTI). These EPBs form post sunset when the solar radiation ceases to ionise the ionosphere. During post-sunset period in the ionosphere, the absence of solar radiations causes rapid decay of the lower ionosphere, and a steep vertical density gradient develops on the bottom side of the raised F-layer.7\n\nThe EPB occurrences may be affected by inter-hemispheric neutral winds (meridional winds) blowing from summer hemisphere to winter hemisphere. Apart from the night-time EPBs occurrences, the daytime occurrence also happens but their cases are very exceptional. The daytime plasma density irregularities is expected to be caused by the irregularities associated with the sporadic E layers. Using the spread-F data has found a small percentage of occurrences of plasma bubbles between sunrise and local noon time.\n\nThe presence of EPBs can degrade the quantity and quality of the user and reference station measurements thereby disrupting the communication from GNSS.8,9 The EPBs can affect the radio-based technologies by scattering and diffracting radio wave signals that pass through them resulting in amplitude and phase scintillations in GNSS signals.5\n\nThere have been many studies on the occurrence characteristics of EPBs using localized observational data such as ionosondes, topside sounders, radio scintillations and in situ measurements.8 A new observational technique of EPBs is ground-based GNSS receiver’s data due to its wide coverage. The time derivative of TEC is often used to measure small-scale ionospheric fluctuations. Time differential TEC is called Rate of TEC (ROT).8 The standard gradient of ROT gives us the Rate of TEC Index (ROTI). In this paper, ROTI is used to identify the occurrence of plasma bubble using the ground-based GPS-TEC data. Hence by using the data from IGS website and by finding the corresponding ROTI values, the EPBs occurrence has been identified.\n\nA solar cycle is the amount of magnetic flux that rises up to the Sun’s surface varies with time in a cycle. This cycle lasts 11 years on average. This cycle is sometimes referred to as the sunspot cycle. Our Sun is a huge ball of electrically-charged hot gas. This charged gas moves, generating a powerful magnetic field. The Sun’s magnetic field goes through a cycle, called the solar cycle. Every 11 years or so, the Sun’s magnetic field completely flips. This means that the Sun’s north and south poles switch places. Then it takes about another 11 years for the Sun’s north and south poles to flip back again. The solar cycle affects activity on the surface of the Sun, such as sunspots which are caused by the Sun’s magnetic fields. As the magnetic fields change, so does the amount of activity on the Sun’s surface. This period of time is considered as cycle 24 which have started in the year 2008 and might end between mid-2019 and late 2020.\n\n\nMethods\n\nGNSS data from IISC station Bangalore, Karnataka, India (latitude- 13.0219°N, longitude-77.5671°E) is collected from SOPAC (SCRIPPS ORBIT AND PERMANENT ARRAY CENTER) archives for the duration 1st January, 2014 to 31st December, 2019 of the 24th solar cycle. SOPAC is a major participant in association with the International GPS Service (IGS), serving as a Global Data Center and a Global Analysis Center. The data is downloaded one day at a time in RINEX (Receiver Independent Exchange) format.10 RINEX is a data interchange format for raw satellite navigation system data in a compressed form. The downloaded RINEX data file is converted to an easily understandable.cmn format using the GPS-TEC application software developed by Dr. Gopi Krishna Seemala.11 The converted data consists of time, PRN, Slant TEC (STEC), Vertical TEC (VTEC), elevation angle and azimuth angle. Only data corresponding to elevation angles greater than 40° were considered as lower elevation angles are associated with increased multipath error. The ROTI based EPB detection algorithm is discussed in section 3.\n\n\nRoti based EPB Detection Algorithm\n\nThe occurrence of EPBs depends on factors such as local time, PRN, Slant TEC (STEC), elevation angle, etc. These parameters refer to occurrence rate, intensity of depletion, depth of depletion and size of EPBs.\n\nTEC can be monitored for identifying possible space weather impacts for the ground to satellite communication and satellite navigation.3 The Rate Of TEC Index (ROTI), defined as the standard deviation of the Rate of change of TEC (ROT). The computation of ROTI involves the time derivative of TEC which automatically eliminates the unknown TEC biases, mitigates the slowly varying background trend of TEC, and emphasizes the high frequency components of TEC fluctuations. ROT is given by equation (1)\n\nSt1and St0are slant TEC at consecutive epoch times t1 and t0, respectively, where t1 > t0.\n\nROTI is given by equation (2).\n\nN is the number of samples.\n\nROT¯=mean of all N ROT values\n\nIn this paper, one 30 min ROTI was derived from the average of six values of 5 min ROTI.8 Daytime ROTI (Rday) is the average value of ROTI computed over a 3 hour time period from 12 hours to 15 hours local time. Evening ROTI (Reve) is the average value of ROTI over a duration of 6 hours from sunset to midnight (i.e., 18 hours to 24 hours) local time. The difference Reve -Rday, on a given day is considered as the decision criterion for EPB detection activity that one day. EPBs are considered to be detected on a given day, if (Reve -Rday) >0.05 TECU/min.12 In this paper, detection of EPBs has been done using ROTI for the waning phase of solar cycle 24.\n\nThis proposed algorithm has been deposited in protocols.io. as “Protocols.io.swapna.karnam”, with the title “Detection of Equatorial Plasma Bubbles”. The detailed protocol with description is available in the following weblink - https://protocols.io/view/detection-of-equatorial-plasma-bubbles-civeue3e with DOI dx.doi.org/10.17504/protocols.io.rm7vzbb12vx1/v1\n\n\nResults\n\nThe processing and analysis of ROTI algorithm was carried out in MATLAB version 2019a software using the tool Research Resource Identifier (RRID) SCR_001622.13\n\nThe data was collected from IGS receiver located at IISC Bangalore, Karnataka, India of year 2016. The time, PRN, STEC, elevation angle were extracted from the collected data. ROTI values were found to detect EPBs as explained in the Methodology section. Figure 1 shows the process flow of the EPB detection algorithm. This study investigates the casual linkage between EPB’s and TEC using solar maximum and minimum period.14\n\nThe analysis of EPBs has been demonstrated for two geomagnetically quiet days and two geomagnetically distributed days. IGS data collected from IISC station on the 6th and 7th February, 2016 were considered to demonstrate the performance of the ROTI based EPB detector for geomagnetically calm days as the average Kp index on those days was 2.\n\nKp is an excellent indicator of disturbances in the Earth’s magnetic field and is used by Space Weather Prediction Centre (SWPC) to decide whether geomagnetic alerts and warnings need to be issued for users who are affected by these disturbances. The Kp-index or Planetary K-index characterizes the magnitude of geomagnetic storms.3 The total day is divided into 8 slots of 3hours each. Figure 2 has been taken from www.magneticstormsonline.com, which depicts the Kp index values on 6th and 7th February, 2016. The average value of Kp index is 2 for both the days indicating geomagnetic calm. Kp index values exceeding 5 indicate a geomagnetic storm.\n\nFigure 3 shows the STEC, ROT and ROTI values on 6th February, 2020 at IISC station for PRN 7. The data from PRN 7 was available from 19.40 hours to 22.40 hours local time. From Figure 3, it can be observed that the STEC values showed a very smooth variation during the entire period with no abrupt variations with a maximum value of 50 TECU. The calculated value of ROT reached a maximum value of 0.6 TECU/min and ROTI values varied from 0 to 0.05 TECU/min. From the smooth curve of STEC and small values of ROTI and ROT it can be hypothesised that 6th February, 2016 was a geomagnetically quiet day.\n\nThe Figure 4 and 5 show the Reve, Rday, (Reve - Rday) values and the threshold of 0.05 TECU/min for 6th and 7th February, 2016 respectively at the IISC station, Bangalore. It can be seen from Figures 4 and 5 that the (Reve -Rday) on both 6th and 7th February, 2016 did not exceed the threshold of 0.05 TECU/min, hence declaring them as EPB free days. The results shown in Figures 4 and 5 are in perfect correlation with the Kp index values of Figure 2.\n\nFigure 6 depicts the Kp index values on 16th and 17th February, 2016 at IISC station, Bangalore. The average value of Kp index on 16th and 17th February, 2016 was found to be 5 which indicates the occurrence of a geomagnetic storm on those two days.\n\nFigure 7 shows the STEC, ROT and ROTI values on 16th February, 2020 at IISC Station, Bangalore from PRN 7. The data from PRN 7 was available from 18.70 hours to 21.70 hours, local time. From Figure 7, it is apparent that the STEC curve depicts ups and downs unlike the smooth curve in Figure 3. The maximum value of STEC recorded on 16th Feb, 2020 was 80 TECU. The STEC value decreased to 0 TECU at 20.40 hours followed by an increase and decrease. The ROT values varied from 0 to 2 TECU/min reaching the maximum at 20.40 hours. The ROTI value reached a maximum of 0.6 TECU/min at 20.40 hours. The variations in STEC, ROT and ROTI during the post sunset hours evidently indicate geomagnetic disturbances on 16th February, 2020 which is in correlation with Figure 6.\n\nFigures 8 and 9 illustrate the Reve, Rday, (Reve - Rday) values along with the threshold of 0.05 TECU/min. It is evident from the Figures 8 and 9 that the Reve values on both 16th and 17th February, 2016 are considerably higher than the Rday values such that the difference (Reve - Rday) exceeded 0.05 TECU/min, thus declaring the presence of EPBs on both the days.\n\nTo study the seasonal EPBs occurrence, the monthly occurrences of 2016 year have been grouped into four seasons namely Vernal/Spring equinox months (February to April), Summer solstice months (May to July), Autumnal equinox months (August to October) and Winter solstice months (November to January).\n\nThe decision criterion (Reve -Rday) values, for the detection of EPBs, for every day of the four seasons, Spring, Summer, Autumn and Winter are shown in Figures 10 to 13 respectively.\n\nIt can be observed from Figure 10, that the number of disturbances in the month of March is more when compared to those in February and April.\n\nFigure 11 shows the days on which EPBs were detected during the summer solstice months of May, June and July. It is apparent from Figure 11 that there were non EPBs detected during the summer solstice months. During these three months, the decision criterion (Reve-Rday) equal to zero.\n\nFigure 12 depicts the (Reve-Rday) values during the Autumnal equinox months of August, September and October. As can be noticed in Figure 12, the number of days on which EPBs were detected is less than those detected during the Vernal equinox months indicating an equinoctial asymmetry.\n\nFigure 13 shows the decision criterion values during the Winter solstice months of November, December and January of the year 2016 at IISC station, Bangalore. From Figure 13, it can be observed that the month of January recorded the maximum number of EPB events in 2016. As per the pre-established EPB climatology, the ionospheric disturbances during equinoctial months are greater than those during the solstice months.15\n\nHowever, an increased number of EPB detections in January indicate a winter anomaly, which also is quite common in low latitude ionosphere.\n\nThe analysis of monthly EPBs occurrences for half of the 24th solar cycle has been carried out, which are shown in Figures 14 to 19. The observed EPBs occurrences were found maximum during month of January, February, March, April, August.\n\nFigure 14 indicates the month wise EPB detection of year 2014 of IISC Bangalore Station. It is observed that the maximum detection of EPBs were in the months of March, February, April and January.\n\nFigure 15 indicates the month wise EPB detection of year 2015 of IISC Bangalore Station. It is observed that the maximum detection of EPBs were in the months of March, April, January, September, August, February, and May.\n\nFigure 16 indicates the month wise EPB detection of year 2016 of IISC Bangalore Station. It is observed that the maximum detection of EPBs were in the months of March, January, February and August.\n\nFigure 17 indicates the month wise EPB detection of year 2017 of IISC Bangalore Station. It is observed that the maximum detection of EPBs were in the months of February, March, April and November.\n\nFigure 18 indicates the month wise EPB detection of year 2018 of IISC Bangalore Station. It is observed that no EPB is detected in this year.\n\nFigure 19 indicates the month wise EPB detection of year 2019 of IISC Bangalore Station. It is observed that October, November and December are the months detected with EPBs.\n\nDuring this half solar cycle period, maximum EPBs occurrences were observed during January. Moreover, the EPBs occurrences during February, March, April and August were also found to be significantly larger than in September, October, November.\n\nThere are no EPBs occurrences for the month May, June, July, December. Occurrence rate of EPBs are expected to be high during the period when the sunset time lag is small, in other words, when the sunsets of the geomagnetic conjugate points are synchronized. This is “magnetic equinox”. The solstice periods are detected with zero and less EPBs. The asymmetric equinox periods are detected with more EPBs. The sunset time lag effect plays an important role for the monthly variation.\n\n\nConclusions\n\nIn this paper the IGS data has been analysed for half solar cycle period from 2014-2019 year for IISC Bangalore, Karnataka, India. The ionospheric errors over low-latitudinal regions has been discussed. The algorithm which can be used to detect the EPBs over low-latitudes has been discussed. The proposed ROTI algorithm detects the occurrence of EPBs using IGS data. Here, we have considered two quiet days and two disturbed days and plotted them. SWPC data is used for comparison of whether geomagnetic alerts and warnings need to be issued for users who are affected by these disturbances and can be concluded that the particular days have geomagnetic disturbances (storms) or not. As the solar cycle is of 11 years, we have considered a half solar cycle for detection of EPBs using ROTI algorithm. The middle years of the solar cycle are detected to have more solar spots and hence years 2014 and 2015 are detected with more no. of EPBs. And the no. of EPBs detected reduced by the end of the solar cycle. This shows that ROTI algorithm is beneficial in detecting EPBs. This ROTI algorithm can be used over GAGAN data to detect EPBs.\n\n\nAuthor contributions\n\nSwapna Raghunath and Ch. Ushakumari: Conception and design of study. Swapna Raghunath, T Padma: Acquisition, analysis and/or interpretation of data. Swapna Raghunath, T Padma, Ch. Usha Kumari: Drafting the manuscript and revising the manuscript critically for important intellectual content.\n\n\nEthical approval and consent\n\nHereby, we Swapna Raghunath and Ch. Usha kumari consciously assure that for the manuscript Equatorial Plasma Bubbles Identification with Single Frequency GNSS Receiver Data is the authors’ own original work, which has not been previously published elsewhere and the paper is not currently being considered for publication elsewhere.",
"appendix": "Data availability\n\nFigshare: [Equatorial Plasma Bubbles Identification with Single Frequency GNSS Receiver]. https://doi.org/10.6084/m9.figshare.21310275.v1\n\nThis project contains the following underlying data:\n\n- Data.zip. The data is considered for finding Equatorial Plasma Bubbles Identification with Single Frequency GNSS Receiver\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).\n\n\nReferences\n\nMannucci AJ, Wilson BD, Yuan DN, et al.: A global mapping techniques for GPS-derived ionospheric total electron content measurements. Radio Sci. 1 May 1998; 33(3): 565–582. Publisher Full Text\n\nFiljar R, Kos T, Markezic I: GPS Ionospheric Error Corrections Modules. IEEE;2006.\n\nSpace Weather Prediction Center: National Oceanic and Atmospheric Administration.Reference Source\n\nRaghunath S, Venkata Ratnam D: Detection of Low-Latitude Ionospheric Irregularities. IEEE Journal of Selected Topics in Applied Earth Observations and Remote Sensing. Nov 2015; 8(11): 5171–5176. Publisher Full Text\n\nShivani B, Raghunath S: Low Latitude Ionosphere Error Correction Algorithms for Global Navigation Satellite System. International Journal of Innvative and Exploring Engineering (IJITEE). 3 January 2020; 9(3): 3244–3248. Publisher Full Text\n\nJiadong S, Yang C, Xie J: China Satellite Navigation Conference (CNSC). Sun JXYJ, editor.Chengdu:Springer Singapore;1 ed.2020; vol. 650. : p. 752.\n\nPanda D, Senapati B, Tyagi B, et al.: Effects of Rayleigh- Taylor instability and ionospheric plasma bubbles on the global navigation satellite system signal. J. Asian Earth Sci. February 2019; 170: 225–233. Publisher Full Text\n\nNishioka M, Saito A, Tsungawa T: Occurrence characterstics of Plasma bubble derived from global ground based GPS receiver networks. Journal of Geophysical Research: Space physics. 02 May 2008; 113(A5): 5–12. Publisher Full Text\n\nKinter M, Beach Paul TL: Simulatenous Global Positioning System observations of equatorial scintillations and total electron content fluctuations. Journal of Geophyscial Research:Space Physics. 01 October 1999; 104(A10): 22553–22565.\n\nBock Y: garner.ucsd.edu/pub/rinex/.2017. [Accessed 23 07 2021].Reference Source\n\nKrishna SG: seemala.blogspot.com/2020/12/gps-tec-program-version-3-for-rinex-3.html.1 Dec 2020. [Accessed 4 Jun 2022].Reference Source\n\nNishoka M, Saito A, Tsugawa T: Occurrence Characteristics of Plasma Bubble Derived from Global Ground-Based GPS Receiver Networks. J. Geophys. Res. Space Physics. May 2008; 113(A05301): 1–12. Publisher Full Text\n\nscicrunch.org/: SciCrunch Infrasructure.[Accessed 5 11 2022].Reference Source\n\nFargundes YPR, Bittencourt JA, Sahai: Transequatrial F-region ionospheric plasma bubbles:solar cycle effects. J. Geophys. Res. Space Physics. February 2000; 62: 1377–1383.\n\nSardul Singh S, Bamgboye DK, McClure JP, et al.: Morphology of equatorial plasma bubbles. J. Geophys. Res. 01 September 1997; 102(A9): 20019–20029. Publisher Full Text"
}
|
[
{
"id": "186600",
"date": "24 Jul 2023",
"name": "Sampad Kumar Panda",
"expertise": [
"Reviewer Expertise Equatorial Plasma bubbles",
"Scintillations",
"Plasma irregularities"
],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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\nThe title “Equatorial Plasma Bubbles Identification with Single Frequency GNSS Receiver Data” is misleading. The authors nowhere used single frequency data for ROTI calculation rather it is dual frequency observables used all throughout the study. I am strongly opposing such a mistake.\nAbstract\nChange “Equatorial and Low latitudinal ionosphere experiences frequent perturbations in plasma density most of which is due to the presence of Equatorial Plasma Bubbles (EPBs).” To “Equatorial and Low latitudinal ionosphere experiences frequent perturbations in plasma density in the post-sunset hours, most of which is due to the presence of Equatorial Plasma Bubbles (EPBs).”\n\nThe statement “EPBs begin to manifest post-sunset and continue to thrive past midnight.” Is wrong. The pre-midnight EPBs are usually more frequent. The post-midnight EPBs are relatively weaker and are expected only during the summer solstice and low solar activity periods.\n\nOmit the sentences “GNSS data has… Exchange (RINEX) format.” and just indicate that the analysis has been done at a low latitude station Bangalore (13.02°N, 77.56°E). Remove the decimal values after second place as you are not going to calculate position here.\n\nI am wondering about any important results kept in the abstract but unfortunately the abstract presents nothing on it.\n\nKey word change from “International GPS Service” “International GNSS Service,”. Moreover, the important key words “scintillation” and “single frequency GNSS” are missing in the keywords. Moreover, where is single frequency data used in this study? After abstract, nowhere it is discussed about single frequency GNSS observation.\n\nIntroduction\nChange the acronym of “International GPS Service (IGS)” as given in my previous comment.\n\n“extending from the receiver to the satellite” It seems the authors are not aware of the GNSS operational mechanism. It should be “extending from the satellite to the receiver”\n\nCorrect the sentence “These EPBs form post sunset when the solar radiation ceases to ionise the ionosphere.”\n\n“The EPB occurrences may be affected by inter-hemispheric neutral winds (meridional winds) blowing from summer hemisphere to winter hemisphere.” Is it the only factor affecting EPBs?\n\nPlease support to the statement with a citation “Using the spread-F data has found a small percentage of occurrences of plasma bubbles between sunrise and local noon time.”\n\nThe authors failed to present introduction section in a sequential manner, swapping between EPBs and scintillations. They should understand that scintillation is seen in radio signals due to EPBs in ionosphere and most part of the F-layer ionosphere corresponds to the total electron content as estimated from GNSS signals. Please rewrite the introduction part with a readable sequence.\n\nCompletely remove the paragraph “A solar cycle is the amount of magnetic flux that rises might end between mid-2019 and late 2020.” as it is irrelevant. You may pick only a sentence or two mentioning the duration considered in the solar activity cycle.\n\nMethods\nRewrite the sentence “GNSS data from IISC station Bangalore, Karnataka, India (latitude- 13.0219°N, longitude-77.5671°E) is collected from SOPAC (SCRIPPS ORBIT AND PERMANENT ARRAY CENTER) archives for the duration 1st January, 2014 to 31st December, 2019 of the 24th solar cycle.” By providing relevant download link, removing the capitalization of words, limiting lat/lon up to 2 significant digits and providing geomagnetic dip or lat/lon of the location for indicating that it is a low latitude station in the Indian longitude sector.\n\n“The difference Reve -Rday, on a given day is considered as the decision criterion for EPB detection activity that one day.” In the introduction, the authors discussed daytime scintillations due to sporadic E. In the given method, how to neglect effects due to possible daytime irregularities as the evening time to daytime subtraction of ROTI may be contaminated with occasional presence of daytime ROTI magnitudes.\n\nRemove the portion “This proposed algorithm has been deposited in protocols.io. as with DOI dx.doi.org/10.17504/protocols.io.rm7vzbb12vx1/v1” as the code is not deposited in the portal along with the algorithm. The steps are already discussed in the paper.\n\nResults\nRemove the portion from the results section and keep them in the methodology part. “The data was collected from IGS receiver EPBs as explained in the Methodology section.\n\nRemove the paragraph “Kp is an excellent indicator of disturbances… indicating geomagnetic calm.” As the content is out of context.\n\n“From the smooth curve of STEC and small values of ROTI and ROT it can be hypothesised that 6th February, 2016 was a geomagnetically quiet day.” This is a wrong conception. Do the authors think that EPBs do not occur on quiet days? Moreover, by taking a single PRN, it is difficult to infer the plasma bubble presence. Mention the azimuth and elevation of the PRN considered to have an exact idea about the signal path.\n\nFigure 4 and 5 are not understandable, all showing straight line, does it mean a single value is considered for each parameter the entire period? Same question for Figures 7 and 8. Please clarify.\n\nProvide the courtesy for the source of the figure borrowed in Figure 2 and 6 including the criteria for considering quiet and disturbed days in the legend.\n\nThe sentence “Figure 6 depicts the Kp index values on 16th and 17th February, 2016 at IISC station, Bangalore.” is wrong. Is the Kp index data taken from any observatory at Bangalore, please understand that it is a global parameter.\n\nBefore the description on Fig. 10, the authors should indicate about the PRNs considered as its preceding study has considered only one PRN.\n\nConclusions\nThe authors implemented the algorithm at a single station, will it be applicable to other low latitude stations ?\n\nRemove the statement “This ROTI algorithm can be used over GAGAN data to detect EPBs.” as it is unrealistic to use ROTI while already high resolution scintillation indices (amplitude and phase) are used in the system and the scale length of irregularities are completely different for scintillation indices and ROTI.\n\nRewrite the conclusion with important results related to 1) using the evening to nighttime subtraction method, 2) its advantages and weakness, 3) diurnal, monthly and seasonal occurrences as observed by the method, 4) comparative statement of the method with other existing methods, 5) a statement saying its substitute role for detecting irregularities in the absence of high resolution scintillation indices.\n\nData availability\nThe authors shared only 6 days data from 2016. By the way the data is freely available to public. What is expected is the authors should share the program developed for detecting EPBs and the final data used for analysis.\n\nAgain I repeat mentioning that work has been conducted using single frequency data is highly questionable as it is clearly understood that the work is done with dual frequency observables.\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?\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": "200563",
"date": "05 Oct 2023",
"name": "Xiaomin Luo",
"expertise": [
"Reviewer Expertise GNSS ionospheric scintillation."
],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 provides an effective method for identifying and correcting EPBs (Equatorial Plasma Bubbles) to improve the positioning and navigation accuracy of GNSS signals. The identification method is proposed based on ROTI (Rate of TEC Index). The authors used GNSS data from Scripps Orbit and Permanent Array Centre (SOPAC). By analyzing six consecutive years of data from 2014 to the second half of the 24th solar cycle in 2019, they found a significant increase in the number of EPBs during the spring and autumn seasons.\nI recommend minor revisions to address some small issues with the text, refinements to the figures, as well as grammar and typographical errors. Overall, the study's findings are valuable, and I recommend that the paper be returned to the authors for minor revisions.\nAbstract\n“Ionosphere is the highest contributor of…”. I'm confused about this sentence. Should the subject be amended to ionospheric scintillation or ionospheric fluctuation? A similar situation is in the second paragraph of Introduction as “Ionosphere is the highest contributor of GNSS positioning error.”.\n\n“In order to improve positional and navigational precision of GNSS signals, EPBs must be identified and corrected.”. EPBs were only identified in the article and not corrected.\n\n“This paper proposes a Rate of TEC Index (ROTI) based EPB identifier using single frequency GNSS receiver data.”. This method appears in reference 12 and is not proposed in this paper, the word propose is not quite accurate, please rephrase it.\n\nIntroduction\nIntroduction section needs to have appropriate references added to the “The EPB occurrences may be affected by…” paragraph and the last paragraph.\n\n“International GPS Service (IGS) is a worldwide network of receivers tracking the transmissions of GNSS satellites is a valuable source of ionospheric data that is globally distributed and continuously available.”. This sentence has too many subordinate parts and contains grammatical mistakes. I suggest revising it to two short sentences that are simple and easy to understand.\n\n“The plasma disturbances in the F-region scatter radio waves and generate rapid fluctuations in the amplitude and phase of GNSS signals called scintillations.”. Ionospheric scintillations occur in more than just the F-region. This sentence needs to be rephrased and the definition of ionospheric scintillation is suggested to be confirmed.\n\n“EPBs are generated through the nonlinear evolution of the Rayleigh-Taylor instability (RTI).”. The sentence you mentioned emphasizes the importance of Rayleigh-Taylor instability for the generation of EPBs. However, in scientific research, there are usually multiple factors and mechanisms that can affect the generation of EPBs, such as temperature and density gradients in the ionosphere, the effects of electric and magnetic fields, solar activity, etc. Therefore, attributing the sole cause of EPB production to the Rayleigh-Taylor instability may be too absolute, as there are other factors that can influence the production of EPBs.\n\nMethods\n“Daytime ROTI (Rday) is the average value of ROTI computed over a 3-hour time period from 12 hours to 15 hours local time. Evening ROTI (Reve) is the average value of ROTI over a duration of 6 hours from sunset to midnight (i.e., 18 hours to 24 hours) local time.” I suppose the article needs to explain why these two time periods were chosen separately for the data calculations. Why couldn't Rday also pick 6 hours to correspond with Reve?\n\nThe first subplot in Figures 3 and 7 shows the variation of STEC. The formula for STEC should be added here to make it easier for the reader to reproduce it.\n\nResults\nFigure 2 and Figure 6 would be more readable with a white background and maintain uniformity throughout the graphic of the article. The grade description in the upper right corner of Figure 2 is not clearly visible. Is it unreasonable to set the horizontal coordinates of the date in the chart, which is usually in the form of Month-Day-Year (MDY)? Figure 2 and 6 used UT but other figures, such as Figure 3, use Local Time. Why not use the same time reference?\n\nFigure 3 is labelled 2020 in the description, but the caption within the figure is 2016, please confirm the exact date of the mapping. Same as Figure 7.\n\nIn Figures 4, 5, 8 and 9, since it is a few straight line ratios, I would recommend adding a table display to label the specific values of the several lines.\n\nFigure 18, where no EPBs were detected, might be considered without this graph. (6) Figures 14 to 19 discuss the month-by-month detection of EPBs from 2014 to 2019, respectively. I suggest placing these figures in a grouping to make comparison easier for the reader. Similarly included are Figures 4 and 5, Figures 8 and 9, Figures 10 to 13.\n\nWhat was the reason for setting up the sub-seasonal experiment and what conclusions were ultimately reached should be explained at the end.\n\nConclusions\n“The middle years of the solar cycle are detected to have more solar spots and hence years 2014 and 2015 are detected with more no. of EPBs.”. The description is not mentioned in the result section. A table summarizing the number of sunspots per year can be added after figures 14 to 19. Or it could be labelled as text in the figure.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Partly\n\nAre sufficient details of methods and analysis provided to allow replication by others? Partly\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nPartly\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Partly",
"responses": []
},
{
"id": "186601",
"date": "05 Oct 2023",
"name": "Lin M.M. Myint",
"expertise": [
"Reviewer Expertise Ionospheric weather",
"equatorial plasma bubbles study",
"GNSS positioning"
],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address 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 would greatly benefit from substantial improvements. Firstly, it needs to improve discussions on the literature review and problem statement. Second, the methodology used in this paper seems to closely resemble the approach presented in reference [8].\nTo outline the paper's contribution, it is required to describe the new aspects of the proposed method or provide a comparison with the exiting work.\n\nImportantly, there are some instances where the interpretation of the data appears to be misleading. Clarity in the explanation of results is important to avoid any misrepresentation.\nThe following are the comments for improving the paper:\n**Abstract:**\nThe statement “In order to improve positional and navigational precision of GNSS signals, EPBs must be identified and corrected.” requires revision for clarity and accuracy.\n\nThe assertion “This paper proposes a Rate of TEC Index (ROTI) based EPB identifier using single frequency GNSS receiver data.” is present, but the single frequency GNSS receiver was not used in this paper.\n\nThe new findings from this work needs to be described.\n\n**Introduction:**\nThe introduction provides fundamental information about the Ionosphere and EPBs which are very generic, it would be beneficial to emphasize the critical aspects related to this study.\n\nWhile several studies have focused on ionospheric irregularities, particularly EPBs, using parameters like TEC and ROTI in regions near the magnetic equator and low latitudes, there is a lack of discussion about prior works in the field of EPB study.\n\nA clear problem statement is lacking in the introduction and it is better to add a comparison between the methodology employed in this paper and that presented in reference [8].\n\n**Results:**\nIt is presumed from Figs 3-5 and 7-9 that the paper employs a single satellite to detect EPBs. A rationale for this selection and the method for choosing a satellite from several visible ones needs to be explained.\n\nThe authors should note the potential occurrence of EPBs during periods of magnetic quietness.\n\nThe statement \" During this half solar cycle period, maximum EPBs occurrences were observed during January\" requires careful reconsideration.\n\nThe statement \"EPB occurrences during February, March, April, and August were significantly higher than in September, October, and November\" needs the author's interpretation.\n\nThe assertion \"There are no EPB occurrences for the months of May, June, July, and December\" contradicts information from Figs 15-19, requiring clarification.\n\nThe sentence \"The asymmetric equinox periods are detected with more EPBs\" needs further clarity.\n\nThe section has numerous figures but lacks sufficient accompanying discussions. The figures should be combined into one. Balancing visuals with comprehensive explanations is essential for a thorough understanding of the data presented.\n\n**Conclusion:**\nThe conclusion should undergo revision to ensure alignment with the content of the preceding sections. For instance, the sentence \"The discussion has centered on ionospheric errors over low-latitudinal regions\" lacks contextual clarity.\n\nThese suggestions aim to enhance the clarity, focus, and cohesiveness of the review comments.\n\nIs the work clearly and accurately presented and does it cite the current literature? No\n\nIs the study design appropriate and is the work technically sound? Partly\n\nAre sufficient details of methods and analysis provided to allow replication by others? No\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\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": []
},
{
"id": "186592",
"date": "18 Sep 2024",
"name": "Alok Taori",
"expertise": [
"Reviewer Expertise Space Sciences",
"Earth & Environmental Sciences",
"Atmospheric Sciences"
],
"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 'Equatorial Plasma Bubbles Identification with Single Frequency GNSS Receiver Data', makes use of a GNSS receiver located at Banglore and identifies the EPBs using TEC variations. Authors use the ROTI index while the world has progressed further at S4 index. TEC measurements with GPS/GNSS receivers are an established method (Briggs B, et al.,1963 [ref 1]; Dasgupta et al., 1983 [ref 2]; Ma G, et al., 2003 [ref 3]; Dashora N, et al., 2005 [ref 4]). The authors need to do a little bit more than these available reports which shall be publishable somewhere.\nThe manuscript has many fundamental glitches. I will iterate only a few of them in the following:\n1) Authors may look into the importance of TEC measurements and the fundamental cause of EPB occurrences. A recently published article by Ghodpage R, et al., 2023 (ref 5) & Patil A. et al. 2023 (ref 6) may benefit them.\n2) Para. 4 discusses EPB & RT instability for which authors presented very fuzzy ideas. The literature followed is also mediocre. Please follow good literature such as Makela J, et al., 2012 (ref 7), Otsuka Y., 2018 (ref 8), Patil A. et al. 2023 (ref 6) which are better and more reasonable review papers and may provide fundamentally correct views on the EPBs and their generation mechanisms.\n3) Para. 8, suggests that 'solar cycle is the amount of magnetic flux that...'. This is wrong. In fact, solar cycle is a representation of convective dynamo action occurring on the sun's surface and the flux tube concept. The sunspots arise due to contrasting luminosity due to these associative phenomena making a solar cycle approximately 11 years. Pipin V. et al., 2012 (ref 9) and Charbonneau P., 2020 (ref 10) explain these processes very nicely.\n\n4) There is absolutely nothing new in EPB detection using ROTI. The detection criteria which they mentioned are much more refined in global scenarios. In fact C/NOFS satellite launched in 2008 already uses a modeled algorithm.\n5) I fail to understand the use of Kp index here. It looks like it is just to state a magnetically disturbed day when Kp is 4!... but, the internationally accepted fact is that it should be more than 5 for moderately disturbed cases. Going further, there is no technical/scientific discussion on the EPB behavior during quiet and disturbed days.\n6) There are too many figures, and discussions are like expanded captions. A reasonable discussion is the soul of any manuscript.\n7) Magnetic equinox hypothesis crosses the scientifically acceptable thresholds. Authors are advised to go through the available literature before such statements.\n\nIs the work clearly and accurately presented and does it cite the current literature? No\n\nIs the study design appropriate and is the work technically sound? Partly\n\nAre sufficient details of methods and analysis provided to allow replication by others? Partly\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\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": []
}
] | 1
|
https://f1000research.com/articles/12-44
|
https://f1000research.com/articles/11-1103/v1
|
27 Sep 22
|
{
"type": "Opinion Article",
"title": "Vaccine Hesitancy in Japan: From a Perspective on Medical Uncertainty and Trans-Scientific Theory",
"authors": [
"Miwako Hosoda"
],
"abstract": "The development and dissemination of vaccines has made immunization possible and has led to the successful control and eradication of various infectious diseases in many parts of the world. However, even when vaccines that are said to be \"effective\" are offered, a certain number of people do not receive them, and this has become a problem known as \"vaccine hesitancy”. Why is vaccine hesitancy a problem? It has been pointed out that the reason is not only because of the risk of contracting infectious diseases if they are not vaccinated, but also because of the lack of a collective immunity system. Vaccines are an effective means of acquiring immunity, but no matter how highly effective vaccines are developed, if the vaccination rate does not exceed a certain number, herd immunity cannot be acquired. Therefore, it is said that how to increase the vaccination rate of the population is a major public health challenge. Hence, the large number of people who do not receive vaccinations due to \"vaccine hesitancy\" is problematic. This paper reviews previous studies on \"vaccine hesitancy\" in Japan and analyzes people's hesitancy in terms of negative \"rumors\" about vaccines, risk perception of vaccine side effects, and sense of burden when receiving vaccinations. Then, I will examine that the background of \"vaccine hesitancy\" is not only distrust of vaccines and risk perception of side effects, but also distrust of those who provide and promote vaccinations, such as medical professionals, government, and public administration. By using medical uncertainty and trans-scientific theory, this paper argues that the problem of \"vaccine hesitancy\" can be reduced if medical professionals and governments show sincere empathy and attitude toward victims of adverse vaccine reactions and those who hesitate to vaccinate.",
"keywords": [
"Vaccine Hesitancy",
"Japan",
"HPV vaccine",
"Uncertainty",
"Trans-Science"
],
"content": "What is vaccine hesitancy?\n\nThe development and widespread use of vaccines has drastically changed the situation in which the only way to prevent infectious diseases was through behavioral changes, such as quarantine. Vaccines have made immunization possible and have been successful in controlling and eradicating various infectious diseases in many parts of the world. However, even when a vaccine that is “scientifically” effective according to peer-reviewed academic papers is offered, a certain number of people do not take the vaccination, and this has become a problem known as “vaccine hesitancy.” It is estimated that 5–10% of the vaccine hesitant population has strong antipathy toward vaccines. It is also clear that there is a significant percentage of people who hesitate to vaccinate even if they do not have such clear intentions.1,2\n\nWhy is “vaccine hesitancy” a problem? It has been pointed out that the reason is not only because of the risk of contracting an infectious disease if they are not vaccinated, but also because of the lack of a herd immunity system. Herd immunity means that by immunizing everyone, it is possible to lower the risk of infection for those in the group who cannot be immunized (e.g., those who are highly susceptible to certain pathogens or allergic).3 The presence of a large number of people in a given population who are immunized against an infectious disease through vaccination reduces the infection rate in the population.\n\nVaccines are an effective means of acquiring immunity. However, no matter how highly effective a vaccine is developed, if the vaccination rate does not exceed a certain number, herd immunity cannot be acquired. Therefore, it is said that how to increase the vaccination rate of a population is a major public health challenge. Therefore, the fact that many people do not receive vaccinations due to “vaccine hesitancy” is problematic.\n\nHow, then, does this “vaccine hesitancy” arise? To preemptively conclude, “vaccine hesitancy” is not caused by distrust of vaccines or the risk of vaccine side effects, as is generally believed, but rather by distrust of the entities that promote vaccination. In other words, people’s distrust of the medical profession, government, and administration is thought to be the cause of their “vaccine hesitancy.” In fact, no matter how much the medical profession, government, and administration scientifically prove the safety of vaccines, and no matter how much they claim that people’s health damage is not caused solely by vaccine side effects, the mindset of those who are “vaccine hesitant” will not change. The aforementioned “vaccine hesitant” people, those who hesitate to vaccinate even if they do not have such clear intentions, do not have a sense of trust in the camps that recommend vaccines: vaccine manufacturers, the medical profession, the Ministry of Health, Labor, and Welfare, and politicians.\n\nThis paper will begin with a review of previous studies in public health and in medicine and health sciences on “vaccine hesitancy” in Japan. By doing so, the author will confirm that most studies analyze people’s hesitancy toward vaccines in terms of negative “rumors” about vaccines, risk perception of vaccine side effects, and sense of burden when receiving vaccinations. Then, by using literature and news reports of patient’s narrative, the author points out that the background of “vaccine hesitancy” is not only distrust of vaccines and risk awareness, but also distrust of those who provide vaccines and promote vaccines, such as medical professionals and the government. Finally, the author suggests that the problem of “vaccine hesitancy” can be reduced if medical professionals and governments show sincere empathy and attitude toward vaccine side effect victims and those who are hesitant to vaccinate.\n\n\nOverview of immunization in Japan\n\nIt is said that vaccine-preventable diseases are best prevented by vaccination. In the past, there was a persistent “rumor” that the measles, mumps, and rubella combined (MMR) vaccine could cause autism. And even now that this has been scientifically proven to be false, “vaccine hesitancy” remains.4 And some parents make the decision not to have their children receive MMR vaccinations. Japan is no exception, and this skepticism about vaccines existed in the past and still exists today.\n\nThe HPV vaccine is intended to prevent cervical cancer, but the HPV vaccination has been a great problem in Japan over the years. In Japan, public subsidies for the HPV vaccine began in 2011, and in 2013, the vaccine became a routine vaccination administered mainly by municipalities based on the law. At that time, girls between the ages of 13 and 16 years were eligible for the public subsidy. According to The Japan Society of Gynecologic Oncology, the HPV vaccination rate was as high as approximately 70% when this system was first introduced.\n\nHowever, adverse events which were called “a variety of symptoms” occurred in girls after HPV vaccination. These symptoms included generalized cramping and the inability to stand or walk after vaccination. These girls were repeatedly reported by the mass media on TV and in newspapers as side effects of the vaccine.5 In addition, a great deal of information about the side effects of HPV was also circulated on social networking services, further increasing the public’s distrust of the vaccine.\n\nAs a result, many girls and their families became hesitant to receive the HPV vaccine. In June 2013, the Ministry of Health, Labor, and Welfare (MHLW) announced that it would no longer actively recommend the HPV vaccine. The MHLW stated, “We will not exclude it from the routine vaccination program. However, it has decided not to actively recommend the vaccine. This was then announced to the general public. The specific wording of the MHLW’s statement at that time was, “We do not actively recommend the vaccination,” and “Please take the vaccination after understanding its benefits and risks.\n\nThis decision by the MHLW led the public to recognize that the government had acknowledged the dangers of the HPV vaccine. Since then, the HPV vaccination rate has plummeted to less than about 1% in just over three years. This withholding has continued for a long time, and by 2020 the vaccination rate had dropped to 0.1%.6 In Europe and the USA, the HPV vaccination rate is between 70% and 80%, so Japan’s low vaccination rate is outstanding.\n\nIt was under these circumstances that the COVID-19 pandemic occurred. Initially, there was no vaccine against COVID-19, and infection was prevented through behavioral measures such as maintaining social distance and wearing masks. However, a vaccine was developed at the end of 2020, making prevention possible. In Japan, a system was prepared for gradual intake starting around the beginning of 2021. The COVID-19 vaccine has been accepted by a relatively large number of the population, with nearly 80% of the population vaccinated for the first and second doses; the third dose is lower, at about 60%, but not lower than in other countries.7\n\nAt the same time, however, many negative “rumors” circulated about the current COVID-19 vaccine, which is a newly developed mRNA-based vaccine. And many people chose not to receive the vaccine. The following is an overview of the reasons for vaccine hesitancy based on previous studies on such “vaccine hesitancy.”\n\n\nHesitations about vaccines 1): negative “rumors” about side effects and vaccination itself\n\nThe media reports of serious side effects, and the spread of such rumors on social networking sites are commonly cited as major reasons for people’s hesitation to get vaccines.8 For example, with regard to the HPV vaccine, “rumors” ran rampant, ranging from “the side effects are so severe that it makes it impossible to lead a daily life,” to “getting the vaccine makes you infertile,” to “the HPV vaccine is a government conspiracy”.\n\nThe following “rumors” about COVID-19 vaccine also spread on SNS: “vaccinations do not work”, “side effects are scary”, “vaccines weaken the human immune system”, and “young women will not be able to have children if they are vaccinated”. Such “rumors” are flying around, and even the opinion that “vaccination is not necessary because antibodies will be produced if the infection occurs naturally” is being circulated. This situation is often evaluated as “low health literacy”.9\n\nHowever, it is also true that some people have suffered serious health problems due to vaccine-induced side effects. For example, even for the COVID-19 vaccine, the MHLW has so far accepted 3,680 applications for relief under the Immunization Health Damage Relief Program.10 And 850 people have been identified as victims of adverse reactions to coronavirus vaccines through the examination. Among them, a woman in her 90s who developed acute allergic reactions and other symptoms after receiving a new coronavirus vaccine and died was included. Thus, it is true that there are health hazards, including deaths, caused by vaccination, so it is necessary to avoid assuming that those who hesitate to be vaccinated are simply influenced by “rumors” in general.\n\n\nHesitancy toward vaccines 2): risk perception toward vaccines\n\nVaccines come with the risk of side effects, and adverse reactions can occur after vaccination. Immunization has the advantage of preventing diseases (benefit), but also has adverse reactions and serious side effects (risk). The Immunization Law in Japan provides for the prevention of infection through the safe administration of vaccinations, as well as for remedial measures in the event of risk. The vaccination system is designed to address unavoidable risks.\n\nPeople with underlying diseases or immune disorders, such as allergies, are considered to be at high risk from vaccinations. Thus, for these people, the risks are higher than the benefits of receiving vaccinations. So, it is precisely because there are potentially people in society who cannot be immunized that it is necessary for those who can be immunized to be vaccinated to prevent infectious diseases. This is the concept of “herd immunity,” which is a basic public health mechanism.11\n\nHowever, if one takes the view that everyone is at risk for vaccination, it becomes difficult to achieve a form of “herd immunity.” This would increase the risk of infection for those who are constitutionally unable to receive vaccinations. Because people have corrected information that vaccinations cannot avoid side effects, they may weigh the benefits of vaccinations against the risks and decide that the risks are greater. Some people try to avoid the risks even when experts agree that the benefits are greater and the risks are smaller. In such cases, it would not be possible to say that such people have low health literacy.\n\n\nVaccine hesitancy 3): burden to vaccinate\n\nRumors and concerns about risks are not the only reasons for vaccine hesitancy. Several studies have shown that the burden of time and cost are the main reasons for “vaccine hesitancy.” For example, there are many types of vaccinations for pediatric infectious diseases. While other countries offer a combination of six vaccines, Japan does not offer simultaneous vaccination with the exception of the MR vaccine. For this reason, parents have to adjust their own schedules and take their children to medical institutions for multiple vaccinations. It has been reported that this high burden for parents leads to “vaccine hesitancy”.12\n\nThe first of these burdens is the time burden.13 In order to receive the vaccine, children must be taken to hospitals and clinics during the daytime when they are open. However, many parents find it difficult to find the time to do so. Often, dual-workers or single-parent families limit the time of day when they can take their children to a medical facility. If they have more than one child, they must ask someone else to babysit, and taking time off work is not an easy task. Second, the vaccine schedule is complicated and burdensome to keep track of.14 However, in Japan, where the practice of having a family doctor is not common, parents are basically responsible for setting up their own vaccine schedule and many parents find this difficult. Third, the number of medical institutions where pediatricians work has been decreasing. In 2008, there were 2906 pediatric facilities, but by 2018, the number had decreased to 2567.15 The resulting decrease in the number of places where vaccinations can be given has also contributed to the difficulties felt by parents. The need for pediatric clinics that are open on weekends has also been pointed out so that parents with weekday jobs can see their children even on weekends.16 Furthermore, many parents feel a financial burden as a fourth factor. The vaccinations themselves are free of charge, as they are covered by public funds. However, the cost of transportation from home to the medical institution, the cost of babysitting if there are other children, and the loss of income due to absence from work are cited as financial burdens. When parents are low-income status, the vaccination rate of their children is markedly lower.17\n\nSuch circumstances that increase the financial burden reflect the current state of Japanese society, in which parents work long hours due to their unstable work style of part-time work and reduced income caused by the recession and divorce. In addition, family members’ circumstances, such as divorce, family discord, and the lack of other reliable guardians due to non-Japanese nationality, are also considered to be causes of “vaccine hesitancy.”\n\n\nChanging treatment of vaccines\n\nAs we have seen above, negative “rumors” about vaccines, concerns about the risk of side effects, and the financial and time burden of vaccinating have been pointed out as reasons for “vaccine hesitancy.” This is also true to some extent for the COVID-19 vaccine. In a study that examined the percentage of Japanese hesitant to receive the COVID-19 vaccine and investigated factors associated with “vaccine hesitancy,” 11.3% (10.9–11.7%) of the 23,142 responses analyzed were hesitant to receive the COVID-19 vaccine.18 This breakdown of “vaccine hesitancy” was higher among young adults and women, particularly among young women (15.6%). On the other hand, the percentage of “vaccine hesitancy” was the lowest among older men, at 4.8%. The most frequently cited reason for not vaccinating was fear of adverse reactions to the vaccine. More than 70% of respondents were concerned about adverse reactions to the vaccine. This percentage of “vaccine hesitancy” toward COVID-19 in Japan was comparable to that of previous studies overseas.\n\nThe age range for COVID-19 vaccination has been gradually lowered in Japan. In conjunction with this, some studies have evaluated factors related to parents’ “vaccine hesitancy” toward COVID-19. An online survey of parents of children aged 3–14 years living in Japan revealed that parents’ “vaccine hesitancy” toward COVID-19 was correlated with the way they obtained information about the vaccine.19 That is, those who cited social media as the most reliable source of information were found to be more hesitant about vaccination than those who trusted official information. Furthermore, more mothers than fathers, and more people with low than high awareness of infection risk, felt “vaccine hesitancy”. The study also showed that those with lower levels of satisfaction with social relationships were more likely to be hesitant to vaccinate.\n\nIn summary, those who are more concerned about the risk of adverse reactions to vaccines, those who get their information from social media, and younger people are particularly hesitant to vaccinate. However, despite this “vaccine hesitancy,” the COVID-19 vaccination rate in Japan is higher than in other countries.\n\nThe Vaccine Confidence Index in Japan is known to be one of the lowest in the world.20 One reason for this is that the MHLW, which promotes immunization, itself had concerns about the safety of the HPV vaccine and stopped actively recommending it in 2013.21 In response to this, Japan’s Ministry of Health and Welfare did what many scientists consider to be a terrible mistake. Some even commented that they were pushed by anti-vaccine activists, who claimed that there were side effects, to stop recommending vaccination to prevent cervical cancer.22\n\nJapan initially approved GlaxoSmithKline’s HPV bivalent vaccine (which protects against the two HPVs with the highest cancer risk) in 2009 and Merck’s quadrivalent vaccine in 2011; in April 2013, the MHLW added both to the national immunization program and began recommending vaccination. However, just 10 weeks later, a number of girls complained of chronic pain, headaches, and motor problems after being vaccinated. The advisory committee therefore proposed to discontinue the recommendation, and the MHLW temporarily suspended the active recommendation in June 2013. As a result, the HPV vaccination coverage plummeted from about 70% to less than 1%, and then to 0.1%.23\n\nHowever, in November 2021, the MHLW announced that it would begin recommending HPV vaccinations in April 2022, on the grounds that the safety of the vaccine had been confirmed. The reason for this is that the HPV vaccination in Japan has been confirmed to be safe. This was based on a campaign to call for vaccination in Japan, as well as academic studies that showed that such safety issues did not appear in clinical trials.24 Another major factor was the announcement by the WHO’s Global Advisory Committee on Vaccine Safety in 2017 that, after an extensive review of studies from around the world, the vaccine was “extremely safe.”25\n\nThe Advisory Committee of the MHLW decided that there was no reason not to resume recommending HPV vaccination.26 In addition, in conjunction with this, the MHLW issued a catch-up vaccination guide in March 2022, for those who missed the HPV vaccination. This refers to a measure that allows women with birthdays between April 2, 1997 and April 1, 2006 who did not receive the HPV vaccine when they should have received it by public expense. Normally, the age range for routine HPV vaccination is from the sixth grade of elementary school to the first grade of high school, that means 12–16 years old. However, as evidenced by the extremely low vaccination rate, many people miss out on HPV vaccination during this period. Therefore, the MHLW decided to offer a new HPV vaccination opportunity for those who are over 16 years old who have not yet been vaccinated. The MHLW has set that period as three years, from April 2022 to March 2025.27 The procedure for receiving the catch-up vaccination is that each eligible person will receive a notice from the municipality in which she has a certificate of residence, which she will take to a medical institution for the vaccination.\n\nThe MHLW has a “Q and A” section on the HPV vaccine on its website. One of the questions is, “Why is there a new vaccination opportunity for women born between 1997 and 2005? The answer to this question is: “Because we are going to offer the opportunity for vaccination to women born between 1997 and 2005. In answer, it says, “From 2013 to 2021, efforts to recommend individual HPV vaccination were withheld. The reason for this, it says, was that “there was a situation in which it was not possible to provide sufficient information on the various symptoms, that could occur after vaccination. It then states that at a meeting of experts in November 2021, it was confirmed that there were no particular concerns about safety and that the effectiveness of vaccination clearly outweighed the risk of adverse reactions, and thus the HPV vaccine was recommended.\n\n\nWhat is vaccine hesitancy?\n\nThe web version of the Yomiuri Shimbun, a national newspaper, introduced the experience of one late teenage college student, A, who became ill after receiving the HPV vaccine.28 A received the first HPV vaccine at the end of her second year of junior high school, and received the A received the first HPV vaccine at the end of her eighth-grade year, and the second in June of her junior year. Two days later, a lump about 3 cm in diameter appeared where she had been vaccinated, and it became painful. When she went to the clinic where she had received the vaccine, the doctor told her that it was a common occurrence. A’s mother was concerned and did some research on the Internet, and found that girls who had been vaccinated against HPV had similar symptoms. Then, she contacted the Association for Victims.\n\nAfter that, A and A’s mother went to the clinic where the vaccine was administered to have a medical certificate written that it was an adverse reaction to the HPV vaccine. However, the doctor at that clinic did not listen to A very much and did not write a diagnosis, saying that he did not think the HPV vaccine was the cause. After that, A visited many hospitals and clinics, but her health showed no sign of getting better.\n\nDuring spring break just before her sophomore year of high school, A went to a reputable osteopathic clinic where many people who had become physically ill after vaccination went. When she told the practitioner at the clinic about her history and symptoms, he listened carefully to her story. This practitioner also palpated the patient and said that it might be an adverse reaction to the vaccine, and told her not to worry because she would recover.\n\nThese words made A feel relieved and happy. She had never had anyone tell her that she would be cured, but this practitioner assured her that she would be cured and did not deny that it was a vaccine side reaction. The practitioner listened carefully to A and A’s mother’s story and said, “I’m sorry you had a hard time.” A’s mother believes that the practitioner expressed hope that she would recover, and that the exercise and diet regimens were effective.\n\nMany healthcare professionals have an attitude of suspicion of “fraud,” refusing to listen to the voices of those who have suffered health problems after receiving vaccines because of the low risk of side effects of vaccines and the scientifically proven fact that vaccines are “extremely safe.” However, this attitude of health professionals creates distrust from the people concerned and society.\n\nIn addition, the author wrote earlier that an expert meeting on the reapproval of the HPV vaccine was held in November 2021. Prior to this meeting, on August 30, 2021, a letter was sent to the Prime Minister, the Chief Cabinet Secretary, and the Minister of Health, Labor and Welfare from the “Diet Members Caucus for the Active Resumption of HPV Vaccine Appreciation.”29 A letter of request was entitled “Request for Prompt Resumption of Active Recommendation of HPV Vaccine.” It stated that since the manufacturers of the HPV vaccine had prepared for a considerable number of inoculations to be available, “it is possible that a situation may arise in which the vaccine that has been prepared may have to be discarded due to expiration of its use”.\n\nWith this, members of the Ombudsperson Conference on Drug Harms have criticized the Diet members for requesting the MHLW to recommend the vaccine in order to contribute to the HPV vaccine manufacturers. Furthermore, the Expert Council of the MHLW has also issued an opinion accusing the MHLW of following the Diet members’ request and deciding to start catch-up vaccinations.30 This also arouses people’s concerns about the government/administration and experts who recommend vaccination. It is possible that people’s “vaccine hesitancy” is caused by the medical profession’s cold attitude toward vaccine victims and distrust of the government and administration, which seem to give priority to the interests of vaccine manufacturers.\n\n\nBeyond the vaccine hesitancy problem\n\nAs we have seen above, the problem of “vaccine hesitancy” is accompanied by reasons that differ from scientific evidence. Therefore, the “vaccine hesitancy” problem is not something that can be resolved by improving people’s health literacy and learning about vaccines correctly, but can be seen as a problem that transcends science. Alvin Weinberg, a nuclear physicist, used the term “trans-science” to indicate that there are social issues that science can ask, but science cannot answer. He wrote as following; “Many of the issues which arise in the course of the interaction between science or technology and society—e.g., the deleterious side effects of technology, or the attempts to deal with social problems through the procedures of science—hang on the answers to questions which can be asked of science and yet which cannot be answered by science.”31\n\nThere are countless questions in medicine that cannot be asked by science. American physician and author Lewis Thomas wrote this about American medicine and society. According to Thomas, the only scientific truth of which we can be entirely confident is that we are utterly ignorant of nature. The sudden confrontation with the depth and scope of “ignorance” is the most important contribution that 20th century science has made to human intelligence.32 Thomas demonstrates the importance of being aware that one cannot be scientifically correct. Regarding the difficulty of judging some things even as an expert, David Bazelon, a judge and jurist, puts it this way. He wrote that in judging factual issues, such as the magnitude of the risks arising from an activity, we as a society should rely on those with the appropriate expertise. However, even in this formulation, many problems remain. There is no clear line between issues of values and issues of fact, and even when properly positioned as issues of scientific fact, there is often no consensus or certainty, even among scientists. Many problems of scientific speculation are in the realm of “trans-science” and cannot be resolved by scientific method and experimentation.33 Medical sociologist Renée Fox has shown in her classic work that medicine is with “uncertainty.”34 Uncertainty has been central to her work since the beginning of medical sociology. Fox then demonstrated the need for careful consideration of the treatment and mishandling of uncertainty and the resulting biohazards, medical risks, and adverse health effects that can result.\n\nIt can be said that the issue of “vaccine hesitancy” is truly a “trans-scientific” issue, and it involves “uncertainty”. How can we approach such a problem that transcends science? It is important for both the medical profession and the government to recognize that not everything is clear, that there are areas of “ignorance,” and that there is “uncertainty” when dealing with vaccines. It is sometimes pointed out that behind the “vaccine hesitancy” is a movement of anti-intellectualism and anti-expertism that has no scientific basis. However, simply viewing “vaccine hesitancy” as “anti-intellectualism” and criticizing it is not the answer to the problem. The reason for “vaccine hesitancy“is not so much a distrust of vaccines per se, but rather a distrust of the regimes that are promoting vaccines: medical, political, and social entities. What can be done to eliminate this distrust? For example, to understand the pain of those who complain of health problems after receiving the vaccines and respond to them with sincerity is necessary. And not call those women who have suffered health problems by vaccination liars, saying that it is not the vaccine’s fault or that they are fraudulent. It is important for experts, governments, and administrations to take the “third way” by accepting the pain of women who have suffered health problems and then advocating for the prevention of cervical cancer. This is true for any vaccine.\n\nKentaro Iwata, a Japanese physician, wrote that “Zero risk is just an impossible illusion”.35 He continued, “It is important to supply vaccines that are safe to some extent and that make more sense than the immediate misery”. In Japan, 3,000 people die each year from cervical cancer, not only because of the low vaccination rate, but also because of the low screening uptake rate. This is also sincerely acknowledged, and the need to consider disease prevention not only with vaccines but also with medical checkups is pointed out. When people’s trust in the medical profession, government, and administration is low, no matter how much the government recommends vaccination, the public will not trust vaccines. In order to solve the problem of “vaccine hesitancy”, it will be essential to build people’s trust in the medical profession, politics, and government, even if this may seem like a roundabout way.\n\n\nData availability\n\nThere are no underlying data associated with this article.",
"appendix": "References\n\nDubé E, Laberge C, Guay M, et al.: Vaccine hesitancy: An overview. Review Hum. Vaccin. Immunother. 2013; 9(8): 1763–1773. PubMed Abstract | Publisher Full Text\n\nMcClure CC, Cataldi JR, OʼLeary ST: Vaccine hesitancy: Where we are and where we are going. Review. Clin. Ther. 2017; 39(8): 1550–1562. PubMed Abstract | Publisher Full Text\n\nFine P, Eames K, Heymann DL: “Herd immunity”: A rough guide. Clin. Infect. Dis. 2011; 52(7): 911916.\n\nDeStefano F, Chen RT: Negative association between MMR and autism. Lancet. 1999; 353(9169): 1987–1988. PubMed Abstract | Publisher Full Text\n\nOkuma Y: Establishment of easy-to-accept screening/Cervical cancer vaccine damage, My Social Security Review, Mainichi Shimbun newspaper, April 10, 2013. (Japanese).\n\nNakagawa S, Ueda Y, Yagi A, et al.: Corrected human papillomavirus vaccination rates for each birth fiscal year in Japan. Cancer Sci. 2020; 111(6): pp. 2156–2162. April 2020. Publisher Full Text\n\nPrime Minister’s Office of Japan, About the new coronavirus vaccine. (Japanese).(Accessed 1 August, 2022).Reference Source\n\nPertwee E, Simas C, Larson HJ: An epidemic of uncertainty: rumors, conspiracy theories and vaccine hesitancy. Nat. Med. 2022; 28: 456–459. Publisher Full Text\n\nBiasio LR: Vaccine hesitancy and health literacy. Hum. Vaccin. Immunother. 2017; 4; 13(3): 701–702. PubMed Abstract | Publisher Full Text\n\nTokyo Shimbun, Health Damage after Corona Vaccination, First relief approved in death case July 25, 2022. (Japanese).(Accessed 1 August, 2022).Reference Source\n\nHosoda M: The story of vaccination that you should know (on-demand edition).2013; Toyo Keizai Shinposha. (Japanese).\n\nUchiyama Y, Katagiri A, Kato H: A Study on the Current Status of Immunization and Parents’ Attitudes and Perceptions, Japan Women’s College of Physical Education. Bulletin of Japan Women’s College of Physical Education. 2012; 42: 1–8. (Japanese).\n\nKawakami Y: Difficulties in autonomous decision-making and informed consent for children’s immunization. Consent in Immunization of Children: A Study of Parents’ Attitudes. Bioethics. 2017; 27(1): 87–95. (Japanese).\n\nMinistry of Health, Labour and Welfare, 2020, Notice on Changes in Vaccination Interval Regulations. (Japanese).(Accessed 1 August, 2022).Reference Source\n\nMinistry of Health, Labour and Welfare, Summary of Medical Facilities (Dynamic) Survey and Hospital Report I, Medical Facilities Survey, 2018. (Japanese).(Accessed 1 August, 2022).Reference Source\n\nOguchi T, Kumakura M: A Survey of Measles and Rubella Vaccination in A Prefecture, Bulletin of the Faculty of Nursing. Dokkyo Medical University;2014; vol. 7. : 1–9. (Japanese)\n\nYamagata C, Haruyama S: Support issues related to child care for needy households with infants and the activities of municipal public health nurses. Journal of Japan Society of Community Health Nursing. 2020; 23(1): 32–41. (Japanese).\n\nOkubo R, Yoshioka T, Ohfuji S, et al.: COVID-19 Vaccine Hesitancy and Its Associated Factors in Japan. Vaccines. 2021; 9(6), 662. (Accessed 1 August, 2022). PubMed Abstract | Publisher Full Text\n\nHoriuchi S, Sakamoto H, Abe SK, et al.: Factors of parental COVID-19 vaccine hesitancy: A cross sectional study in Japan. PLoS One. 2021; 16(12): e0261121. (Accessed 1 August, 2022). PubMed Abstract | Publisher Full Text\n\nde Figueiredo A , Simas C, Karafillakis E, et al.: Mapping global trends in vaccine confidence and investigating barriers to vaccine uptake: A large-scale retrospective temporal modelling study. Lancet. 2020; 396: 898–908. PubMed Abstract | Publisher Full Text\n\nSimms KT, Hanley SJB, Smith MA, et al.: Impact of HPV vaccine hesitancy on cervical cancer in Japan: A modelling study. Lancet Public Health. 2020; 5: e223–e234. PubMed Abstract | Publisher Full Text\n\nNormile D: Japan relaunches its HPV vaccination drive. For thousands of women, Science Insider Asia/Pacific, 29 MAR 2022.(Accessed 1 August, 2022).Reference Source\n\nMinistry of Health, Labor and Welfare, Know about HPV Vaccine, Cervical Cancer Prevention Frontline. (Japanese).(Accessed 1 August, 2022).Reference Source\n\nSuzuki S, Hosono A: No association between HPV vaccine and reported post-vaccination symptoms in Japanese young women: Results of the Nagoya study. Papillomavirus Res. 2018; 5: 96–103. PubMed Abstract | Publisher Full Text\n\nWorld Health Organization: Meeting of the Global Advisory Committee on Vaccine Safety, 7–8 June 2017. Wkly Epidemiol. Rec. 2017; 92: 393–402. (Accessed 1 August, 2022). PubMed Abstract Reference Source\n\nMinistry of Health, Labour and Welfare, Human Papillomavirus Infection - Cervical Cancer (Uterine Cancer) and HPV Vaccine. (Japanese).(Accessed 1 August, 2022).Reference Source\n\nMinistry of Health, Labour and Welfare, HPV Catch-up Vaccination. (Japanese).(Accessed 1 August, 2022).Reference Source\n\nIwanaga N: Cervical Cancer Vaccine Special - From the Standpoint of a Woman in Her Late Teens and Her Mother Who Suffered Physical Problems After Vaccination, Yomi Doctor, The Yomiuri Shimbun, November 8, 2016. (Japanese).(Accessed 1 August, 2022).Reference Source\n\nDiet Members Caucus for Resuming Active Appreciation of HPV Vaccine, Request for Prompt Resumption of Active Recommendation of HPV Vaccine, dated August 30, 2021. (yesJapanese).\n\nKumamoto K: I Don’t Want a Council of Hide-and-Seek, Responsibilities of Experts and Media, Enishi wo Kizuna Kai, Crosstalk, July 31, 2022. (Japanese).\n\nWeinberg A: Science and Trans-Science. Minerva. 1974; 10(2): 209–222. Publisher Full Text\n\nThomas L: The Medusa and the Snail. N. Engl. J. Med. 1977; 296: 1103–1105. PubMed Abstract | Publisher Full Text\n\nBazelon DL: Risk and Responsibility. Science. 1979; 205: 277–280. Publisher Full Text\n\nFox RC: The Evolution of Medical Uncertainty. Milbank Memorial Fund Quarterly/Health and Society. 1980; 58(1): 1–49. Publisher Full Text\n\nIwata K: Do vaccinations “work”? Considering Vaccine Haters, Kobunsha Shinsho; 2010. (Japanese)."
}
|
[
{
"id": "151664",
"date": "11 Oct 2022",
"name": "Carrie Mae Long",
"expertise": [
"Reviewer Expertise Immunology",
"vaccinology",
"bacteriology."
],
"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 submitted manuscript aims to describe vaccine hesitancy in Japan and provide recommendations for the reduction of this phenomenon based on the concepts of “medical uncertainty” and “trans-scientific theory”. The author suggests that empathetic attitudes shown by governmental and medical professionals are a key consideration in addressing vaccine hesitancy. The manuscript addresses an important, timely topic and provides valuable insight. Indeed, a growing body of literature addressing this theme exist; however, I feel that the submitted manuscript does not adequately capture this breadth. Furthermore, the author’s arguments were not consistently supported by the published literature. I feel that the manuscript’s clarity and organization could be improved. Major and minor comments are as follows:\nMajor Comments:\nI felt that the manuscript could be more concisely written, allowing for the inclusion of more prescient information. This was especially apparent in the abstract, which I feel could be shortened and more focused. Further, the general organization of the manuscript could potentially be improved by renaming select section headers (e.g. “Overview of immunization in Japan” to “Overview of vaccine hesitancy in Japan”; There are two sections named “What is vaccine hesitancy?”).\n\nAt times, the author’s statements were not supported by citations. For example: “It is estimated that 5-10% of the vaccine hesitant population has strong antipathy…”; “To preemptively conclude, “vaccine hesitancy” is not caused by distrust of vaccines or the risk of vaccine side effects, as is generally believed, but rather by distrust of the medical…”. Further, the author states that “no matter how much the medical profession, government, and administration scientifically prove the safety of vaccines, and no matter how much they claim that people’s health damage is not caused solely by vaccine side effects, the mindset of those who are “vaccine hesitant” will not change.” There are numerous studies that suggest the opposite1-4. In addition, there are studies addressing the author’s core argument of the importance of empathy and I feel that these studies should be cited 5-9. Overall, I feel that amelioration of the lack of relevant citations and ensuing discussion would strengthen the author’s arguments and the manuscript as a whole.\n\nI feel that the concepts of “medical uncertainty” and “trans-scientific theory” should be defined earlier in the manuscript and described more comprehensively, given their importance to the author’s central argument.\n\nMinor Comments:\nI felt that the use of quotation marks was not consistent. Perhaps this could be addressed for clarity.\n\nThe statement that “People with underlying diseases or immune disorders, such as allergies, are considered to by at high risk from vaccinations.” is not necessarily true for all vaccines. I suggest clarifying this statement as most contraindications are vaccine/platform specific.\n\nIs the “MR” vaccine reference in the “Vaccine hesitancy 3): burden to vaccinate” section referring to the “MMR” vaccine?\n\nIs the topic of the opinion article discussed accurately in the context of the current literature? Partly\n\nAre all factual statements correct and adequately supported by citations? Partly\n\nAre arguments sufficiently supported by evidence from the published literature? Partly\n\nAre the conclusions drawn balanced and justified on the basis of the presented arguments? Partly",
"responses": [
{
"c_id": "9131",
"date": "11 Jan 2023",
"name": "Miwako Hosoda",
"role": "Author Response",
"response": "Dear Dr. Carrie Mae Long, Thank you very much for your review. I learned a lot from your suggestion. I would like to answer each of your comments. Comment 1: I felt that the manuscript could be more concisely written, allowing for the inclusion of more prescient information. This was especially apparent in the abstract, which I feel could be shortened and more focused. Further, the general organization of the manuscript could potentially be improved by renaming select section headers (e.g. “Overview of immunization in Japan” to “Overview of vaccine hesitancy in Japan”; There are two sections named “What is vaccine hesitancy?”). My answer 1: The abstract is within the designated word count, so I think it is fine as it is, but I have shortened it a bit according to your advice. I changed the section headers of second “What is vaccine hesitancy?” to \"Social Structure that causes vaccine hesitation\" Comment 2: At times, the author’s statements were not supported by citations. For example: “It is estimated that 5-10% of the vaccine hesitant population has strong antipathy…”; “To preemptively conclude, “vaccine hesitancy” is not caused by distrust of vaccines or the risk of vaccine side effects, as is generally believed, but rather by distrust of the medical…”. Further, the author states that “no matter how much the medical profession, government, and administration scientifically prove the safety of vaccines, and no matter how much they claim that people’s health damage is not caused solely by vaccine side effects, the mindset of those who are “vaccine hesitant” will not change.” There are numerous studies that suggest the opposite1-4. In addition, there are studies addressing the author’s core argument of the importance of empathy and I feel that these studies should be cited 5-9. Overall, I feel that amelioration of the lack of relevant citations and ensuing discussion would strengthen the author’s arguments and the manuscript as a whole. My answer 2: In order to be more accurate in content, the text has been rewritten as follows. And added supportive references. “no matter how much the medical profession, government, and administration scientifically prove the safety of vaccines, and no matter how much they claim that people’s health damage is not caused solely by vaccine side effects, it may change the mindset of those who are at some vaccine hesitant, but it cannot reduce it to zero.” Comment 3: I feel that the concepts of “medical uncertainty” and “trans-scientific theory” should be defined earlier in the manuscript and described more comprehensively, given their importance to the author’s central argument. My answer 3: I add the concepts of “medical uncertainty” and “trans-scientific theory” earlier in the manuscript and described more comprehensively. Comment 4: I felt that the use of quotation marks was not consistent. Perhaps this could be addressed for clarity. My answer 4: I have standardized the use of quotation marks. Comment 5: The statement that “People with underlying diseases or immune disorders, such as allergies, are considered to by at high risk from vaccinations.” is not necessarily true for all vaccines. I suggest clarifying this statement as most contraindications are vaccine/platform specific. My answer 5: I changed the sentence. “People with underlying diseases or immune disorders, such as allergies, are mostly considered to be at high risk from vaccinations.” Comment 6: Is the “MR” vaccine reference in the “Vaccine hesitancy 3): burden to vaccinate” section referring to the “MMR” vaccine? My answer 6: In many other countries, the MMR vaccine is used, but in Japan, the MMR vaccine is not used; the MR vaccine is used. I have added important references. If you have any further questions, please let me know. Thank you again for your effort. Best regards, Miwako Hosoda"
}
]
},
{
"id": "151669",
"date": "22 Nov 2022",
"name": "Nelson Filice de Barros",
"expertise": [
"Reviewer Expertise Sociology of Health and Public 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\nProfessor Hosoda’s paper reviews studies on \"vaccine hesitancy\" in Japan, discuss this issue with trans-scientific theory, and points that the \"vaccine hesitancy\" can be reduced if medical professionals and governments show sincere empathy and attitude toward victims of adverse vaccine reactions and those who hesitate to vaccinate.\nAs Professor Hosoda affirms, “the reason for “vaccine hesitancy“ is not so much a distrust of vaccines per se, but rather a distrust of the regimes that are promoting vaccines: medical, political, and social entities”. They present a very important, contemporary, event related to the HPV vaccination in Japan and its relationship with “vaccine hesitancy” in Japan and other places. To Professor Hosoda, the hesitations about vaccines are related to: negative “rumors” about side effects and vaccination itself, risk perception toward vaccines and burden to vaccinate.\nResistance to vaccination has been present in different countries since the second half of XIX Century1-3 and, certainly, it is related to the COVID-19 anti-vaccination movement. However, to better understand the “vaccine hesitance” related to the COVID-19 pandemic Professor Hosoda could introduce in their paper the recent debate about the intellectual property of the COVID-19 vaccines and the \"apartheid of vaccines\".\nThe “vaccine hesitancy”, for Professor Hosoda, is co-related it with trans-scientific theory. They assume, as Alvin Weinberg, that there are “questions which can be asked of science and yet which cannot be answered by science”. However, it could be positive to expand the trans-scientific theory with the biopolitics perspective, from Michel Foucault.4,5 The hesitations about vaccines are less related to the science issues and more associated to the political issues, because the “the quest for certainty in science is ongoing”6, but many times the science consensus, by definition very different from truth, has been used as unquestionable truth, and serving as social technology of control.\nProfessor Hosoda finalises the paper affirming that “it will be essential to build people’s trust in the medical profession, politics, and government”. Their position is very close to the idea of the “century of the patient”7, which states that “the health care system inherited from the 20th century falls short on doctors and patients. (…) The 21st century should become the century of the patient (…) centered around patients—not industries, organizations, or doctors”. Considering the proximity of their ideas it could be important whether they discuss some of the seven “sins” the authors developed.\nAlthough I have suggested expanding the discussion with \"apartheid of vaccines\" and biopolitics, this does not mean that the article is superficial in relation to the debate on \"vaccine hesitancy\", on the contrary, it is a very representative text on the issue of vaccination in Japan.\n\nIs the topic of the opinion article discussed accurately in the context of the current literature? Yes\n\nAre all factual statements correct and adequately supported by citations? Partly\n\nAre arguments sufficiently supported by evidence from the published literature? Yes\n\nAre the conclusions drawn balanced and justified on the basis of the presented arguments? Yes",
"responses": [
{
"c_id": "9132",
"date": "11 Jan 2023",
"name": "Miwako Hosoda",
"role": "Author Response",
"response": "Dear Dr. Nelson Filice de Barros, Thank you very much for your review. I learned a lot from your comments. I update the current version of manuscript by responding the other reviewers' comments. Thank you again for your contribution. Best regards, Miwako"
}
]
},
{
"id": "153930",
"date": "13 Dec 2022",
"name": "Tomoko Steen",
"expertise": [
"Reviewer Expertise Population genetics",
"epidemiology",
"microbiome",
"immune system",
"gut-brain axes",
"biomedical science policy and advocacy",
"and clinical pharmacology."
],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nGeneral Comment:\nProfessor Hosoda has done substantial research on the topic through a literature search on relevant articles and websites. Many of the statements here are backed up with appropriate references, but some are missing. I discuss them below.\nIt is shocking that Japan, a homogenous country with obedient populations, is facing substantial vaccine hesitation. The proposal to improve Japanese people’s health literacy, explaining the link between “public health and vaccine,” as well as improving communication between medical professionals and the public, might be one of the only solutions to reduce vaccine hesitancy in Japan. Yet, this is a complex issue globally. It is not only communication between the public and health care providers or public health literacy. Much of this is a political belief held by some individuals. I argue this because many healthcare providers are hesitating to take vaccines. Vaccine hesitancy is a serious public health concern and a complex issue to solve. The situation is more significant in the United States, where political issues participate in public health decisions. Until I read Professor Hosoda’s article, I was not fully aware of the extent of vaccine hesitancies in Japan. However, on one occasion, I was shocked to hear that a Japanese colleague (a daughter of a leading medical school professor in Japan) said that they hesitated to obtain the vaccine because of a severe side effect reported. If the vaccine’s side effects happen to you or your loved ones, it is 100% risk; an individualistic country such as the United States would be the first to refuse vaccines if higher stakes are reported while your mission is to take a vaccine for the common good.\nVaccine hesitancy should not be a political issue, but it is in many countries. Overall, Professor Hosoda’s analysis is highly informative, and the article should be indexed after minor revisions.\nProfessor Hosoda’s substantial case studies are quite impressive. I recommend indexing after a minor proof read, and revisions.\n\nSpecific Comments:\nSome critical information or logical argument needs to be included in some areas.\nPage 1 For example, in the “Abstract,” “Therefore, it is said that how to increase the vaccination rate of the population is a major public health challenge. Hence, the substantial number of people who do not receive vaccinations due to \"vaccine hesitancy\" is problematic.” This is a confusing sentence: The author needs to explain “herd immunity” before these sentences, then the section after these sentences makes sense.\n\nPage 1 I also do not necessarily fully agree with the author’s conclusion here: “By using medical uncertainty and trans-scientific theory, this paper argues that the problem of \"vaccine hesitancy\" can be reduced if medical professionals and governments show sincere empathy and attitude toward victims of adverse vaccine reactions and those who hesitate to vaccinate.” I think education/health literacy and “mutual aid”/familiar good spirit are more important than promoting empathy in one group of people.\n\nPage 3 The author needs to add a citation here: “According to The Japan Society of Gynecologic Oncology, the HPV vaccination rate was as high as approximately 70% when this system was first introduced.”\n\nPage 3 The author did not mean “girls” but rather “symptoms of girls” or “side effects girls experience” in the sentence here. “The mass media repeatedly reported these girls on TV and in newspapers as side effects of the vaccine.”\n\nPage 3 You meant “the side effects of HPV (missing the word “vaccine”)” here. “In addition, a great deal of information about the side effects of HPV was circulated on social networking services, further increasing the public’s distrust of the vaccine.”\n\nPage 3 “This is the concept of ‘herd immunity,’ which is a basic public health mechanism” No definition of proper “herd immunity” is in the article; I suggest adding the following:\nA proper definition of Herd Immunity: - Herd immunity is reached when a sufficient proportion of the community in a defined area develops a threshold level of immunity to new infection that prevents exponential spread of infections in that community”. For COVID-19, the effective reproduction number [R0], the number of community members infected by each case of virus in question, has been used as reference. This number has to be below one to avoid spread1-4.\n\nPage 4: Besides reference 10 of Tokyo Shinbun, the author should check the governmental information at the COVID-19 site on this issue should cite.\n\nIs the topic of the opinion article discussed accurately in the context of the current literature? Yes\n\nAre all factual statements correct and adequately supported by citations? Yes\n\nAre arguments sufficiently supported by evidence from the published literature? Yes\n\nAre the conclusions drawn balanced and justified on the basis of the presented arguments? Yes",
"responses": [
{
"c_id": "9133",
"date": "11 Jan 2023",
"name": "Miwako Hosoda",
"role": "Author Response",
"response": "Dear Prof. Tomoko Steen Thank you very much for your review. I learned a lot from your suggestion. I would like to answer each of your comments. Specific Comments: 1.Page 1 For example, in the “Abstract,” “Therefore, it is said that how to increase the vaccination rate of the population is a major public health challenge. Hence, the substantial number of people who do not receive vaccinations due to \"vaccine hesitancy\" is problematic.” This is a confusing sentence: The author needs to explain “herd immunity” before these sentences, then the section after these sentences makes sense. Response 1 I added the following explanation in the earlier point of the paper with the references. \"Herd immunity means that the risk of person-to-person transmission is reduced when a significant portion of the population becomes immune to an infectious disease. While it is not appropriate to overemphasize the benefits of herd immunity, it is certainly possible to confirm its substantial effectiveness.\" Anderson R.M., May R.M. Vaccination and herd immunity to infectious diseases. Nature. 1985;318:323–329. Randolph HE, Barreiro LB. Herd Immunity: Understanding COVID-19. Immunity. 2020 May 19;52(5):737-741. doi: 10.1016/j.immuni.2020.04.012. PMID: 32433946; PMCID: 2.Page 1 I also do not necessarily fully agree with the author’s conclusion here: “By using medical uncertainty and trans-scientific theory, this paper argues that the problem of \"vaccine hesitancy\" can be reduced if medical professionals and governments show sincere empathy and attitude toward victims of adverse vaccine reactions and those who hesitate to vaccinate.” I think education/health literacy and “mutual aid”/familiar good spirit are more important than promoting empathy in one group of people. Response 2 Thank you for pointing this out. For the sake of accuracy, I have made the following changes. \"Various approaches have been pointed out to reduce vaccine hesitancy, including education, health literacy, and consideration about individual stress level.\" Biasio LR. Vaccine hesitancy and health literacy. Hum Vaccin Immunother. 2017 Mar 4;13(3):701-702. doi: 10.1080/21645515.2016.1243633. Epub 2016 Nov 3. PMID: 27808587; PMCID: PMC5360145. Zhang H, Li Y, Peng S, Jiang Y, Jin H, Zhang F. The effect of health literacy on COVID-19 vaccine hesitancy among community population in China: The moderating role of stress. Vaccine. 2022 Jul 30;40(32):4473-4478. doi: 10.1016/j.vaccine.2022.06.015. Epub 2022 Jun 8. PMID: 35710509; PMCID: PMC9174466. 3. Page 3 The author needs to add a citation here: “According to The Japan Society of Gynecologic Oncology, the HPV vaccination rate was as high as approximately 70% when this system was first introduced.” Response 3 I added the following papers as reference. Sekine M, Kudo R, Yamaguchi M, Hanley SJB, Hara M, Adachi S, Ueda Y, Miyagi E, Ikeda S, Yagi A, Enomoto T. Japan's Ongoing Crisis on HPV Vaccination. Vaccines (Basel). 2020 Jul 6;8(3):362. doi: 10.3390/vaccines8030362. PMID: 32640691; PMCID: PMC7565470. Kudo R, Yamaguchi M, Sekine M, Adachi S, Ueda Y, Miyagi E, Hara M, Hanley SJB, Enomoto T. Bivalent Human Papillomavirus Vaccine Effectiveness in a Japanese Population: High Vaccine-Type-Specific Effectiveness and Evidence of Cross-Protection. J Infect Dis. 2019 Jan 9;219(3):382-390. doi: 10.1093/infdis/jiy516. PMID: 30299519; PMCID: PMC6325350. 4. Page 3 The author did not mean “girls” but rather “symptoms of girls” or “side effects girls experience” in the sentence here. “The mass media repeatedly reported these girls on TV and in newspapers as side effects of the vaccine.” Response 4 I changed as follows. “The mass media repeatedly reported these symptoms of girls on TV and in newspapers as side effects of the vaccine.” 5. Page 3 You meant “the side effects of HPV (missing the word “vaccine”)” here. “In addition, a great deal of information about the side effects of HPV was circulated on social networking services, further increasing the public’s distrust of the vaccine.” Response 5 I added \"vaccine\" after HPV. “In addition, a great deal of information about the side effects of HPV vaccine was circulated on social networking services, further increasing the public’s distrust of the vaccine.” 6. Page 3 “This is the concept of ‘herd immunity,’ which is a basic public health mechanism” No definition of proper “herd immunity” is in the article; I suggest adding the following: A proper definition of Herd Immunity: - Herd immunity is reached when a sufficient proportion of the community in a defined area develops a threshold level of immunity to new infection that prevents exponential spread of infections in that community”. For COVID-19, the effective reproduction number [R0], the number of community members infected by each case of virus in question, has been used as reference. This number has to be below one to avoid spread1-4. Response 6 I added the explanation of \"herd immunity\" by following the advice. \"Herd immunity is reached when a sufficient proportion of the community in a defined area develops a threshold level of immunity to new infection that prevents exponential spread of infections in that community”. For COVID-19, the effective reproduction number [R0], the number of community members infected by each case of virus in question, has been used as reference. This number has to be below one to avoid spread1-4.\" References 1. Desai A, Majumder M: What Is Herd Immunity?. JAMA. 2020; 324 (20). Publisher Full Text 2. Randolph HE, Barreiro LB: Herd Immunity: Understanding COVID-19.Immunity. 2020; 52 (5): 737-741 PubMed Abstract | Publisher Full Text 3. Delamater PL, Street EJ, Leslie TF, Yang YT, et al.: Complexity of the Basic Reproduction Number (R0).Emerg Infect Dis. 2019; 25 (1): 1-4 PubMed Abstract | Publisher Full Text 4. Barker P, Hartley D, Beck AF, Oliver GH, et al.: Rethinking Herd Immunity: Managingthe Covid-19 Pandemic in a DynamicBiological and Behavioral Environment. NEJM Catalyst. 2021. Reference Source 7. Page 4: Besides reference 10 of Tokyo Shinbun, the author should check the governmental information at the COVID-19 site on this issue should cite. Response 7. I added a report of MHLW which shows a mechanism of the side effect victims. The exact number of the people who got side effect was only announced to the mass media, so I keep putting the reference of Tokyo shinbun. Ministry of Health, Labour and Welfare, Health Risk Relief for Novel Coronavirus Vaccine Infectious Disease and Immunization Review Subcommittee Meeting December 9, 2021, 145th Disease and Disability Certification Examination Committee, December 9, 2021. (Japanese) Thank you again for your careful review. If you have any questions and/or concerns, don't hesitate to contact with me. Best regards, Miwako Hosoda"
}
]
}
] | 1
|
https://f1000research.com/articles/11-1103
|
https://f1000research.com/articles/12-36/v1
|
10 Jan 23
|
{
"type": "Research Article",
"title": "Anti-inflammatory effect of omega-7 against doxorubicin-induced cardiotoxicity in male rats: An observational study",
"authors": [
"Mohammed H. Fadhel",
"Ali Faris Hassan",
"Ali Faris Hassan"
],
"abstract": "Background: Doxorubicin is a crucial anticancer medication, however, cardiotoxicity is a severe adverse effect of doxorubicin therapy. Various mechanisms, including inflammation, have been postulated to account for this negative effect. The omega-7 fatty acid is a polyunsaturated fatty acid with anti-inflammatory and antioxidant properties. Therefore, the study's objective was to see whether omega-7 had any possible protective benefits against doxorubicin-induced cardiotoxicity in male rats. Methods: 28 male Wister rats were split into four groups (seven per group). Group 1 (negative control): healthy animals received normal saline orally as the vehicle for eight successive days and were sacrificed on day nine. Group 2 (positive control): animals that received a single dose of doxorubicin hydrochloride (IP 15mg/kg) were sacrificed the next day. Group 3: the animals were administered omega-7 orally at a 100 mg/kg/day dose for eight days. A single injection of doxorubicin IP (15 mg/kg) was given on day nine. The animals were sacrificed on day 10. Group 4: the animal was administered omega-7 orally at a 300 mg/kg/day dose for eight days. A single injection of doxorubicin IP (15 mg/kg) was given on day nine. The animals were sacrificed on day 10. Serum was collected and used to measure lactate dehydrogenase, creatinine kinase-MB, tumor necrosis factor-alpha, interleukin-6, and interleukin-1beta. Results: Lactate dehydrogenase and creatinine kinase-MB levels in group 3 (100mg/kg) were significantly lower than in group 2 (p>0.05) and significantly lower in group 4 (300mg/kg) than in group 2. Tumor necrosis factor-alpha, interleukin-6, and interleukin-1beta levels were considerably lower in the omega-7-treated groups (100 and 300mg/kg) than in the positive control group (p<0.05). Conclusion: Through a mechanism involving the reduction of inflammation, omega-7 may preserve the cardiac tissue against doxorubicin-induced damage.",
"keywords": [
"Doxorubicin",
"Omega-7",
"lactate dehydrogenase",
"Tumor necrosis factor-alpha",
"Interleukin-6."
],
"content": "Introduction\n\nDoxorubicin is a highly effective chemotherapeutic agent used to treat several types of cancer, such as hematogenous and solid cancers.1 Unfortunately, this medication's clinical utility is constrained due to its adverse effects, particularly cardiotoxicity, which may include cardiac arrhythmias, congestive heart failure, left ventricular dysfunction, and cardiomyopathy.2 It has been suggested that oxidative stress, apoptosis, autophagy, and Endoplasmic reticulum (ER) stress are the mediators of Doxorubicin-induced cardiac damage.3 Doxorubicin-induced cardiac damage is also influenced by inflammation. It has been demonstrated that doxorubicin therapy significantly increases the expression of tumor necrosis factor-alpha (TNFα), interleukin (IL)-1β, and IL-6.4 In addition, phosphokinase C (PKC) has been suggested to act as an intracellular signaling mediator for TNFα activity, which causes cytotoxicity and apoptosis in various cell types.5 The membranes of cells and organelles are usually permeable to fatty acids, and the administration of unsaturated fatty acids such as omega-3, -6, and -9 fatty acids, or various combinations of these fatty acids, has been studied for its potential health benefits.6 It has also been suggested that omega-7 fatty acid supplements may affect serum inflammatory biomarkers.6 The primary components of omega-7 are palmitoleic acid (16:1, cis-9-hexadecenoic acid) and vaccenic acid [(11E)-11-octadecenoic acid] ERER, which are primarily found in cold-water fish and sea buckthorn berries.7 Omega-7 is a non-essential fatty acid in humans as it can be produced endogenously.8 The consumption of omega-7 fatty acids has been related to better health outcomes by increasing HDL cholesterol levels and decreasing LDL cholesterol levels.7 Earlier research has demonstrated that omega-7 fatty acids efficiently suppress H2O2-induced inflammatory factors such as prostaglandin E2 (PGE2), tumor necrosis factor- (TNF-α), and interleukin-1 (IL-1β).8 Nevertheless, the effects of omega-7 on the integrity of cardiac tissue are yet to be established. As a result, we want to look into whether omega-7 may prevent against doxorubicin-induced cardiotoxicity by reducing inflammation.\n\n\nMethods\n\nThe investigations were conducted in accordance with the criteria set by the University of Baghdad, College of Pharmacy Ethics Committee, with permission number 490, on February 9, 2022. The animal investigation was conducted in compliance with the ARRIVE principles 2.0 and the preclinical ARRIVE Essential 10 checklist.9 In addition, every effort was made to make rats as comfortable as possible during tests and sampling.\n\nAn observational study was done employing a convenient sample of male rats from 1/4/2022 to 30/6/2022.\n\nIn this investigation, the medications doxorubicin hydrochloride and omega-7 were utilized. Doxorubicin was obtained from Pfizer Laboratories, New York, United States, with the batch number FE0746, and omega-7 was purchased from Source Naturals, United States, Batch number SN 2552.\n\nTwenty-eight male Wister rats weighing 150 and 250 grams were kept in polypropylene cages under regulated circumstances, including a regular light/dark cycle and a temperature of 22 2 °C. Rats were given commercial pellets and water from the tap.\n\nThe rats were split into four groups using simple random selection; including seven rats in each group as the following:\n\n1. Group 1 (negative control): healthy animals received normal saline orally as the vehicle for eight successive days and were sacrificed on day nine.\n\n2. Group 2 (positive control): Animals that received a single dose of doxorubicin hydrochloride (IP 15 milligrams/kilogram) and were sacrificed the next day.\n\n3. Group 3: the animals were administered omega-7 orally at a 100 milligrams/kilogram/day dose for eight days. A single injection of doxorubicin IP (15 milligrams/kilogram) was given on day nine. The animals were sacrificed on day 10.\n\n4. Group 4: the animal was administered omega-7 orally at a 300 milligrams/kilogram/day dose for eight days. A single injection of doxorubicin IP (15 milligrams/kilogram) was given on day nine. The animals were sacrificed on day 10.\n\nTwenty-four hours after the last administration of the drug, gel-activated tubes were used to collect blood samples after they were drawn from the carotid artery and let to stand for 30 minutes to clot. Then, the blood was spun in a centrifuge for 15 minutes at 3000 rpm to gain the serum.10 The collected serum was used to determine the cardiac biomarkers (LDH and CK-MB) and the pro-inflammatory mediators (TNFα, IL-6, and IL-1β). Bioassay Technology Laboratory, China provided all ELISA kits; details are presented in Table 1 below.\n\nVersion 27 of IBM SPSS Statistics (RRID: SCR 016479) for Windows Operating System was utilized all through the statistical analysis method. The mean and standard deviation of all study data were reported (SD). The Shapiro-Wilk test was used to determine if the findings were normal. Furthermore, an unpaired T-test was used to determine the significance of the difference in means between two independent samples. P values less than 0.05 were considered statistically significant.\n\n\nResults\n\nAnalysis of the data in Table 1 revealed a significant increase in lactate dehydrogenase (LDH) levels in group 2 (positive control) compared to group 1 (negative control) at p < 0.05. Despite the absence of a significant difference p > 0.05 in the level of LDH in group 3 (100 mg) when compared to group 2, the level of LDH decreased significantly p < 0.05 in group 4 (300 mg/kg omega-7) when compared to group 2. The level of creatinine kinase-MB (CK-MB) was significantly elevated in group 2 (positive control) compared to group 1 (p < 0.05). While there was no significant difference in the level of CK-MB between groups 3 (100 mg) and 2 (p > 0.05), the higher dose of omega-7 (300 mg/kg) in group 4 significantly decreased the level of CK-MB compared to group 2 (p < 0.05; Table 2).\n\n* Significantly different compared to group I (negative control) (p < 0.05).\n\n# Significantly different compared to group II (positive control) (p < 0.05).\n\nThe serum concentration of TNF-α was substantially elevated in the doxorubicin-treated group compared with the control group (p < 0.05; Figure 1). In contrast, the co-administration of omega-7 in groups 3 (100 mg/kg) and 4 (300 mg/kg) decreased TNF-α levels considerably compared to group 2 (positive control) p < 0.05. IL-6 levels were considerably elevated in group 2 (positive control) compared to group 1 (negative control) in Figure 2 (p < 0.05). In contrast, the co-administration of omega-7 in groups 3 (100 mg/kg) and 4 (300 mg/kg) substantially decreased IL-6 levels compared to group 2 (positive control) p < 0.05. Significantly more IL-1 was detected in group 2 (positive control) than in group 1 (positive control), p < 0.05. As demonstrated in Figure 3, the co-administration of omega 7 (100 mg/kg) in group 3 and (300 mg/kg) in group 4 decreased IL-1 considerably p < 0.05 compared to group 2 (positive control).\n\n\nDiscussion\n\nThe high prevalence of cardiomyopathy and heart failure is the main side effect of doxorubicin usage.11 According to several investigations, the production of reactive oxygen species (ROS) from doxorubicin redox activation contributes significantly to the cytotoxicity of doxorubicin.11 Numerous other studies have concentrated on the signaling mechanism that causes doxorubicin-induced apoptosis.12,13 Moreover, pro-inflammatory cytokines such as IL-6, TNFα, and IL-1β are implicated in activating inflammatory reactions.14 It has previously been noted that doxorubicin causes heart tissue to release more pro-inflammatory cytokines.15 In the present study, the administration of doxorubicin to rats resulted in cardiotoxicity, as evidenced by significant increases in LDH and CK-MB serum levels (Table 1), which have been utilized in various preclinical investigations to evaluate heart injury,16,17 combined with a significant increase of serum levels of pro-inflammatory cytokines including TNFα, IL-1β, and IL-6; similar to other findings.15,18\n\nPre-treatment with omega-7 in group 3 (100 mg/kg) caused a non-significant difference in LDH and CK-MB levels compared to the positive control group. However, pre-treatment with a larger dose (300 mg/kg) showed a significant reduction in LDH and CK-MB levels compared to group 2. This outcome is in line with earlier investigations into the effectiveness of polyunsaturated fatty acid as a cardioprotective subsistence.19\n\nTNFα is an inflammatory cytokine produced by macrophages and monocytes during acute inflammation and is in charge of several cell-signaling processes that result in necrosis or apoptosis.20 From the study, the level of TNFα was significantly reduced in the omega-7 pretreated group (100 mg and 300 mg/kg), which is in line with other research about the anti-inflammatory effect of omega-7 in H2O2-treated cells.8 Interleukin-6 (IL-6) is known to regulate immune responses and is considered a potentially helpful indicator of immune system activity.21 Inflammation, infection, and cardiovascular disease may cause an increase in IL-6. IL-1β is a cytokine that plays a role in pain, inflammation, and autoimmune diseases.22 From the study, the levels of both IL-6 and IL-1β were significantly reduced in omega-7 treated groups (100, 300 mg/kg) compared to the positive control group. The result is consistent with another published study on omega-3 concerning those biomarkers.23 However, the current study is the first to demonstrate the efficacy of omega-7 in lowering the elevated levels of IL -6 and IL -1β associated with doxorubicin-induced cardiotoxicity. The production of several cytokines, including TNFα, during the oxidative stress brought on by the doxorubicin therapy, served as a mediator for cytotoxicity.24 It was discovered that omega-7, which was used in this study to protect the myocardial tissue from doxorubicin toxicity, inhibited the excessive release of TNFα through a mechanism linked to the suppression of the PKC enzyme responsible for the production of NF-kβ, the crucial signaling molecule in the pathway that releases TNFα.25 Therefore, an appealing treatment strategy for treating doxorubicin-induced cardiotoxicity is to modify the expression of the cytokines.26 The presentation of TNF and other inflammatory mediators during oxidative stress situations is inhibited by substances like omega-7, which may have potential therapeutic utility in this area.\n\n\nConclusion\n\nThe current discovery suggests that omega-7 may shield the myocardium against doxorubicin-induced damage by reducing the inflammatory response.",
"appendix": "Data availability\n\nZenodo: Measurement of TNF-α, CK-MB, LDH, IL-1 and IL-6 levels for the anti-inflammatory effect of omega-7 against doxorubicin-induced cardiotoxicity in male rats: an observational study. https://doi.org/10.5281/zenodo.7353404. 27\n\nThis project contains the following underlying data:\n\n‐ Article data.xlsx (Measurement of TNF-α, CK-MB, LDH, IL-1 and IL-6 levels for the anti-inflammatory effect of omega-7 against doxorubicin-induced cardiotoxicity in male rats: an observational study).\n\nZenodo: The ARRIVE Essential 10: author checklist – Anti-inflammatory effect of omega-7 against doxorubicin-induced cardiotoxicity in male rats: an observational study. https://doi.org/10.5281/zenodo.7360390. 9\n\n- The ARRIVE Essential 10: author checklist.pdf\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).\n\n\nReferences\n\nGianni L, Myers CE: The role of free radical formation in the cardiotoxicity of anthracycline. Cancer Treatment and the Heart. 1992; 9–46.\n\nCarvalho C, Santos RX, Cardoso S, et al.: Doxorubicin: the good, the bad and the ugly effect. Current Medicinal Chemistry. 2009; 16(25): 3267–3285. PubMed Abstract | Publisher Full Text\n\nRidha DKA, Al-Shawi NN: The Impacts of Graded Doses of Pyridoxine on the Biomarkers, Aspartate Aminotransferase, lactate Dehydrogenase and Total Antioxidant Capacity in Doxorubicin-Induced Cardiotoxicity in Female Rats. Iraqi Journal of Pharmaceutical Sciences. 2017; 12–21. (P-ISSN: 1683-3597, E-ISSN: 2521-3512). Publisher Full Text\n\nYarmohammadi F, Karbasforooshan H, Hayes AW, et al.: Inflammation suppression in doxorubicin-induced cardiotoxicity: natural compounds as therapeutic options. Naunyn-Schmiedeberg's Archives of Pharmacology. 2021; 394(10): 2003–2011. PubMed Abstract | Publisher Full Text\n\nAbdulrazzaq MH: Protective effect of benfotiamine against doxorubicin-induced cardiotoxicity in rabbits. Iraqi Journal of Pharmaceutical Sciences. 2007; 16(1): 14–17. (P-ISSN: 1683-3597, E-ISSN: 2521-3512). Publisher Full Text\n\nSasagawa M, Boclair MJ, Amieux PS: Omega-7 Mixed Fatty Acid Supplementation Fails to Reduce Serum Inflammatory Biomarkers: A Placebo-Controlled, Double-Blind Randomized Crossover Trial. Nutrients. 2021; 13(8): 2801. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMehta P, McAuley DF, Brown M, et al.: COVID-19: consider cytokine storm syndromes and immunosuppression. Lancet (North American ed). 2020; 395(10229): 1033–1034. Publisher Full Text\n\nSong I-B, Gu H, Han H-J, et al.: Effects of 7-MEGA™ 500 on oxidative stress, inflammation, and skin regeneration in H2O2-treated skin cells. Toxicological Research. 2018; 34(2): 103–110. PubMed Abstract | Publisher Full Text | Free Full Text\n\nFadhel MH:The ARRIVE Essential 10: author checklist – Anti-inflammatory effect of omega-7 against doxorubicin-induced cardiotoxicity in male rats: an observational study. [Data set]. Zenodo. 2022. Publisher Full Text\n\nAbd MR, Hassan AF: The Ameliorative Effect of Fimasartan against Methotrexate-Induced Nephrotoxicity in Rats. Iraqi Journal of Pharmaceutical Sciences. 2022; 31(1): 87–94. (P-ISSN 1683-3597 E-ISSN 2521-3512). Publisher Full Text\n\nBuzdar AU, Marcus C, Blumenschein GR, et al.: Early and delayed clinical cardiotoxicity of doxorubicin. Cancer. 1985; 55(12): 2761–2765. PubMed Abstract | Publisher Full Text\n\nSawyer DB, Fukazawa R, Arstall MA, et al.: Daunorubicin-induced apoptosis in rat cardiac myocytes is inhibited by dexrazoxane. Circulation Research. 1999; 84(3): 257–265. PubMed Abstract | Publisher Full Text\n\nNakamura T, Ueda Y, Juan Y, et al.: Fas-mediated apoptosis in adriamycin-induced cardiomyopathy in rats: in vivo study. Circulation. 2000; 102(5): 572–578. PubMed Abstract | Publisher Full Text\n\nAlbensi BC: What is nuclear factor kappa B (NF-κB) doing in and to the mitochondrion? Frontiers in Cell and Developmental Biology. 2019; 7: 154. PubMed Abstract | Publisher Full Text | Free Full Text\n\nBirari L, Wagh S, Patil KR, et al.: Aloin alleviates doxorubicin-induced cardiotoxicity in rats by abrogating oxidative stress and pro-inflammatory cytokines. Cancer Chemotherapy and Pharmacology. 2020; 86(3): 419–426. PubMed Abstract | Publisher Full Text\n\nSheibani M, Nezamoleslami S, Faghir-Ghanesefat H, et al.: Cardioprotective effects of dapsone against doxorubicin-induced cardiotoxicity in rats. Cancer Chemotherapy and Pharmacology. 2020; 85(3): 563–571. PubMed Abstract | Publisher Full Text\n\nYagmurca M, Fadillioglu E, Erdogan H, et al.: Erdosteine prevents doxorubicin-induced cardiotoxicity in rats. Pharmacological Research. 2003; 48(4): 377–382. PubMed Abstract | Publisher Full Text\n\nBenzer F, Kandemir FM, Kucukler S, et al.: Chemoprotective effects of curcumin on doxorubicin-induced nephrotoxicity in wistar rats: by modulating inflammatory cytokines, apoptosis, oxidative stress and oxidative DNA damage. Archives of Physiology and Biochemistry. 2018; 124(5): 448–457. PubMed Abstract | Publisher Full Text\n\nEl Amrousy D, El-Afify D, Khedr R, et al.: Omega 3 fatty acids can reduce early doxorubicin-induced cardiotoxicity in children with acute lymphoblastic leukemia. Pediatric Blood Cancer. 2022; 69(7): e29496. PubMed Abstract | Publisher Full Text\n\nIdriss HT, Naismith JH: TNFα and the TNF receptor superfamily: Structure-function relationship(s). Microscopy Research and Technique. 2000; 50(3): 184–195. PubMed Abstract | Publisher Full Text\n\nJordan SC, Choi J, Kim I, et al.: Interleukin-6, a cytokine critical to mediation of inflammation, autoimmunity and allograft rejection: therapeutic implications of IL-6 receptor blockade. Transplantation. 2017; 101(1): 32–44. PubMed Abstract | Publisher Full Text\n\nKendall H, Marshall R, Bartold P: Nitric oxide and tissue destruction. Oral Dis. 2001; 7(1): 2–10. Publisher Full Text\n\nOrchard TS, Gaudier-Diaz MM, Phuwamongkolwiwat-Chu P, et al.: Low sucrose, omega-3 enriched diet has region-specific effects on neuroinflammation and synaptic function markers in a mouse model of doxorubicin-based chemotherapy.2018; 10(12): 2004.\n\nMeldrum DR, Cleveland JC Jr, Cain BS, et al.: Increased myocardial tumor necrosis factor-α in a crystalloid-perfused model of cardiac ischemia-reperfusion injury. The Annals of Thoracic Surgery. 1998; 65(2): 439–443. PubMed Abstract | Publisher Full Text\n\nMoscat J, Diaz-Meco MT, Rennert P: NF-κB activation by protein kinase C isoforms and B-cell function. EMBO Reports. 2003; 4(1): 31–36. PubMed Abstract | Publisher Full Text | Free Full Text\n\nBarnes PJ, Karin M: Nuclear factor-κB—a pivotal transcription factor in chronic inflammatory diseases. New England Journal of Medicine. 1997; 336(15): 1066–1071. Publisher Full Text\n\nFadhel MH:Measurement of TNF-α, CK-MB, LDH, IL-1 and IL-6 levels for anti-inflammatory effect of omega-7 against doxorubicin-induced cardiotoxicity in male rats: an observational study. [Data set]. Zenodo. 2022. Publisher Full Text"
}
|
[
{
"id": "160118",
"date": "27 Jan 2023",
"name": "Laith G. Shareef",
"expertise": [
"Reviewer Expertise clinical chemistry",
"clinical pharmacy"
],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 well-written, addresses a core problem (doxorubicin-induced cardiotoxicity), and provides evidence about the efficacy of omega-7 fatty acids as a preventative agent. I have the following comments/suggestions for the author’s consideration:\nAlthough the mechanism of cardio-toxicity is adequately outlined in the introduction section, it would be preferable to explain it a little more, as Doxorubicin administration increases oxidative stress due to the production of free radicals and causes a cardiac deficit of antioxidants. At the nuclear level, intercalation into DNA, leading to inhibition of synthesis of macromolecules, has been suggested, which precipitates DNA abnormalities like alkylation, crosslinking, strand separation, and helicase activity. DNA damage may be initiated via topoisomerase II inhibition and apoptosis induction in response to topoisomerase II inhibition [1].\n\nThank you for including information on laboratory animals in the materials and methods section. However, could you further explain other facts, such as where these animals were obtained?\n\nWas the humidity level recorded? (If yes, please provide the relative humidity level).\n\nYou indicated that these animals were provided with a regular light/dark cycle; please describe how this was accomplished (for how many hours per day for each mode).\n\nCould you briefly describe the \"Survival study\" on mortality and general conditions?\n\nWithin the \"Experimental design\" section, you stated that the rats were split into four groups using simple random selection, including seven in each group from groups 2 through 4. You mentioned the dose of doxorubicin hydrochloride of 15mg/kg; how did this dose arrive at? Please explain with a citation.\n\nWithin group 2, doxorubicin hydrochloride was administered as a single dose; in which route of administration? Please clarify.\n\nWith groups 3 and 4, you mentioned that omega-7 administered as 100 milligrams/kilogram/day and 300 milligrams/kilogram/day, respectively. Please clarify how these doses were determined, with reference.\n\nDescribe any efforts to address potential sources of bias.\n\nWas any other software besides SPSS employed to present the data for the statistical analysis? For example, GraphPad Prism for Windows? Please declare\n\nIt is essential to compare the following parameters LDH, CK-MB, TNF, IL-1, and IL-6 after doxorubicin administration to their baseline levels rather than the control group alone.\n\nHow many animals died before the termination of the experiment in the doxorubicin groups?\n\nAny documented overall weight reduction with doxorubicin treatment over time? Any cardiac weight loss? These symptoms may be suggestive of cardiotoxicity and a loss of appetite, as well as a deterioration in heart function that leads to a lower cardiac weight-to-body weight ratio and increased mortality.\n\nIs the work 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/12-36
|
https://f1000research.com/articles/12-35/v1
|
10 Jan 23
|
{
"type": "Research Article",
"title": "The average age of first marriage for Indonesian women in their reproductive period who give birth to an average of two children: National survey (2017 – 2019)",
"authors": [
"Mario Ekoriano",
"Muthmainnah Muthmainnah",
"Anastasia Titisari",
"Yuli Puspita Devi",
"Teguh Widodo",
"Edy Purwoko",
"Mario Ekoriano",
"Anastasia Titisari",
"Yuli Puspita Devi",
"Teguh Widodo",
"Edy Purwoko"
],
"abstract": "Background: The results of the 2020 population census in Indonesia showed that the population has reached 270.020.000 million. This number shows that the population in Indonesia during the last 10 years has increased by around 32,56 million people. One of the BKKBN's efforts to reduce the birth rate in Indonesia is to urge people to marry at the ideal age, 21 years for women and 25 years for men.\n\nMethods: This study used GPAS/ SKAP data for the period 2017, 2018, and 2019 using the module for women aged (15-49) and the sample used was all women aged 40-49. This analysis used secondary data from the 2017, 2018, and 2019 government performance and accountability surveys. Results: The age at first marriage for women should be encouraged to be 25 years old, not 21 years old, which has always been echoed and socialized. Age at first marriage is the most dominant factor in women aged (40-49) to have more than two children (2017 SOR: 4.17 95% CI [1.85-17.31], (2018 SOR: 57.14 99% [4.12-793.67]) (2019 SOR 21.22 99% CI [2.28-197.45]), while only in 2019 the AFM variable after controlling for it remained significant in influencing having children more than 2 (AOR 27.64 99% [2.88-265.20]).\n\nConclusion: Women aged (40-49) who have a younger age at first marriage (10-24) have a longer reproductive age range, so they have relatively more children than women who married at the age of 25 and over. After controlling for other factors, the characteristics of women who have a tendency to have more than two children. Therefore, it is necessary to design a health strategy that is more suitable to the needs, characteristics, and capacity of women to reduce the birth rate in Indonesia is to urge people to marry at the ideal age.",
"keywords": [
"age first marriage",
"women",
"reproductive period",
"early marriage",
"ideal family size",
"national survey",
"good health",
"well-being"
],
"content": "Introduction\n\nThe population census in 2020 in Indonesia showed the total population has reached 270,02 million. This figure, of course, shows that the total population in Indonesia over the last 10 years has increased by around 32.56 million people.1 The increasing number of Indonesians has made the government through the National Population and Family Planning Board (BKKBN) always try to reduce the total fertility rate (TFR) for women aged 15-49 years by setting the TFR to 2.1 in 2024. Various efforts have been made by government, such as increasing the coverage of modern contraceptive use, marrying at the ideal age and providing family planning socialization to the community through IEC (Information, Education, and Communication).2\n\nOne of the BKKBN’s efforts to reduce the birth rate in Indonesia is to urge people to marry at the ideal age, 21 years for women and 25 years for men.3 Maturation of the age of marriage is not only beneficial for the health of women’s reproductive organs, but also shortens the reproductive period of women, which begins at the age of 15 and ends on average at the age of 49 years. Their reproductive years peak at around the age of 20 and decline gradually in their 30s. Women in their 30s who are healthy and fertile have a 20 percent chance of getting pregnant. However, for women who are 40 years old, the chance of getting pregnant is only 5 percent every month.4 Research conducted by Adhikari in Nepal at 2010 also explained that the age at first marriage of a woman significantly significantly effects the average number of children born in Nepal. Women who marry younger will give birth more children until the end of their reproductive age (40-49).\n\nMeanwhile, the results of the government performance and accountability survey [GPAS/SKAP] from 2016 to 2019 show the trend of the average age at first marriage in Indonesia ranging from 20 to 21 years and the total fertility rate [TFR] from 2016 to 2019 tends to increase. There is no significant change in this figure to achieve the TFR target of 2.1 in 2024. Based on the explanation above, it is necessary to know what is the right age at first marriage for women so that they give birth to an average of 2 children and what factors affected these births over the 3 survey periods. This study also explored the age at first marriage in each island in Indonesia, which of course is also important to know because it has different characteristics.\n\n\nMethods\n\nThis study was a quantitative study with cross-sectional design. This study used secondary data which was Government Performance and Accountability Survey data 2017-2019. The data had been collected for three months (July to October) in each year.\n\nThe GPAS/SKAP survey in 2017 to 2019 had a three-step sampling framework. The first stage of sampling used a list of villages or sub-districts throughout Indonesia complete with urban/rural classifications. The second sampling framework produced a list of clusters in the selected villages, while the third stage took a sample of selected households based on the household listings made by the enumerator by conducting door-to-door data collection in the selected cluster.\n\nThe GPAS/SKAP survey enumeration is a cluster consisting of several nearby census blocks consisting of approximately 200 households. Stratified multistage random sampling was used as the overall sampling strategy. A total of 1912 clusters were selected from 2017 to 2018 and there were an additional 23 clusters to 1,935 villages in 2019 in Indonesia (spread across 34 provinces and 514 districts). Villages were clusters which constitute the enumeration area and have been allocated to each province based on urban and rural strata using the proportional probability sample method and sizes based on socio-economic background (rural-urban and wealth index) in various regions.\n\nThen a number of 35 households were selected in each cluster using a systematic random sampling technique which was carried out randomly on a list of all households (listing) by the enumerator/interviewer in the selected cluster, resulting in a total of 66,920 households [GPAS/ SKAP 2017-2018] and 67,725 households [GPAS/SKAP 2019].\n\nA questionnaire trial was also conducted prior to data collection to assess the extent to which participants were able to answer the list of questions, and to observe how the questions matched the smartphone application. The practice of recording households was carried out during the trial to determine the number of households in the selected cluster that could be used as a reference in data collection. The practice of recording is also intended so that interviewers/enumerators can understand the recording mechanism and the ideal number of interviewers when taking notes in the field.\n\nThis survey has 4 four modules, namely the household module, the family module, the module for women aged [15-49] years old and the module for single adolescents age [15-24] years. Data collection was conducted digitally to ensure quality and so the data analysis process could be carried out faster. The enumerator/interviewer was equipped with a mobile phone and the Mobile Collection application (developed from Open Data Kit) includes all modules digitally and these are connected online to a server. Total GPAS/ SKAP of women aged (15-49) in 2017 was 52,340 [15-49] with the unit of analysis 15,978 women aged [40-49]; GPAS/ SKAP of women aged (15-49) 2018 totaled 60,599 [15-49] with a unit of analysis of 17,463 women aged [40-49]; GPAS/SKAP of women aged (15-49) 2019 totaled 59,824 [15-49] with a unit of analysis of 18,007 women aged [40-49].\n\nThis research has obtained approval from the Ethics Review Team through a letter from the Head of the Ethics Review Team number 1281/PD.101/H4/2018 and 454/LB.02/H4/2019 and the survey implementation is based on the regulation of the Head of BKKBN number 11/2018. This study obtained the informed written consent from participants by hardcopy and digital were available in Android which only can be accessed by the enumerators, supervisors and central data management. The participants were told that their data would be used and published aggregately.\n\nData analysis used normalization weighting for each level in the province aggregated from SKAP/GPAS Report 2019. This study used descriptive and inferential analysis. Descriptive analysis aimed to determine the average age at first marriage by region, while inferential analysis, logistic regression was used (with controlling variables such as education, wealth index, residence, and occupation) to test and determine the most influential variables. So that the data can be used as a basis for making future policies.\n\n\nResults\n\nBased on Tables 1, 2 and 3, it can be seen that when women in the category of age at first marriage [AFM] are 10-14 years old, they have 3 to 4 children on average. The results of the GPAS/SKAP from 2017 to 2019 showedwomen who are married between 10-14 years tend to experience a decrease in births, but still have an average of 3 to 4 children. The highest fertility pattern [of children ever born in ALH] by region for AFM 10-14 years occurred in Maluku Island, namely 5.17 in 2017, then increased in 2018 [5.81] and decreased in 2019 to 4.76. The lowest birth rates in AFM [10-14] years occurred in Java and Bali, namely 3.50 in 2017, experienced a slight increase to 3.52 in 2018 and decreased to 3.19 in 2019.\n\nWhile women in the AFM category [15-19] years in total, the average birth rate was still at 3.52 in 2017 and continued to decline to 3.02 in 2019. Although there was a downward trend in the number of children born each year, Figure 1 still shows that the average number of children born is 3. Meanwhile, based on archipelagic areas, AFM [15-19] years in Maluku Island has the highest birth rate, namely 4.46 in 2017, experiencing a fairly high decline to 4.29 in 2018 and then rising again in 2019 to an average 4.52 children. The lowest birth rate by region at the age of [15-19] years occurred in Java and Bali, which was around 2.97 in 2017 and continued to decline to an average birth of 2.78 children in 2019.\n\nSource: GPAS/SKAP of 2017, 2018, and 2019.\n\nThen, the AFM for women aged 20-24 years old continued to decline to 3.06 in 2017 and also in 2018 and 2019 to 2.66. By region, the island of Maluku is still the region with the highest birth rate, namely 3.87 in 2017. This condition decreased to an average birth rate of 3.76 and then rose again to 3.84 in 2018-2019 respectively. The lowest birth rate for women with AFM [20-24] still occurs in Java and Bali, namely 2.55 which then decreased to 2.01 in 2018. However, this condition increased again in 2019 with an average birth rate of to 2.44.\n\nBased on the results of the analysis of the 3 year survey data period above, the birth rate with an average of 2 children based on the table above can be achieved when AFM occurs in the [25-29] year category, namely 2.30 in 2017, which continues to experience a decrease to 2.17 in 2018 and slightly increase again in 2.22. The highest birth rate by region at the time of AFM [25-29] was still outperformed by Java and Bali, namely 1.97; 2.01 from 2017-2018 and slightly increased in 2019 to 2.07. Meanwhile, the highest birth rate by region is still occupied by Maluku Island, which is 2.76 years. 2017, then experienced an increase of 3.14 in 2018 then fell back to an average of 3.03 in 2019.\n\nThe older a woman’s AFM, the lower the birth rate. This is shown by AFM in the age category 30-34 in 2017-2019, women had given birth to children under 2 children (2.05 to 1.60). The average of women who gave birth consistently decreased from period AFM of 30-34 until the end of their reproductive period AFM of 40-44 category. This indicates that the older a person gets married for the first time, the more difficult it is to have children.\n\nOverall, the highest birth rate is on the island of Maluku with an average birth of 3.80 children in 2017, then in 2018 it increased to an average of 3.66 children then increased again to an average of 3.78 children in 2019. Meanwhile, the lowest birth rates occurred in Java and Bali, reaching an average of 2.64 children in 2017, then increasing to an average of 2.55 children in 2018 and to an average of 2.48 in 2019.\n\nTable 4 shows a comparison of factors related to giving birth to more than two children in 2017-2019 in women of childbearing age (aged 15-49), where it can be seen that the characteristics of women who have a tendency to have more than two children, are namely: area of residence, education level, economic status, working status, preference for the ideal number of children more than two, AFM, and family planning methods used. Economic status, age at first marriage, and use of contraceptives were significant factors influencing women aged 15-49 to have more than 2 children in the 3 survey periods [2017, 2018 and 2019] even after controlling for other factors. This shows that the 3 factors need the government’s attention on women in order to control or plan the number of children they give birth to.\n\n* Significant 0.1.\n\n** Significant 0.05.\n\n*** Significant 0.01.\n\nFrom 2017 to 2019, age at first marriage was the most dominant factor for women aged 15-49 to have more than two children (2017 SOR: 4.17 95% CI [1.85-17.31], (2018 SOR: 57.14 99% [4.12-793.67]), (2019 SOR 21.22 99% CI [2.28-197.45]). While in 2019, the AFM variable for the age category 10-14 which after being adjusted by other variables remained significant in influencing women having more than 2 children (AOR 27.64 99% [2.88-265.20]). For women aged 15-49, more than 2 children is the ideal number desired, namely in 2018 (AOR: 6.55 99% CI [5.97-7.19] and in 2019 (AOR 6.58 99% [6.11-7.08]).\n\n\nDiscussion\n\nMarriage is a complex life stage. It also has an impact on the demographics and health of both individuals and communities related to the time of delivery.5,6 Early marriage can have a negative impact on maternal and child health, including malnutrition and increased morbidity and mortality.5 Delaying the age of marriage to a certain age can have a positive impact for women as it gives them time to develop and actualize themselves,7 as well as their reproductive health because if the marriage age is too young, the reproductive organs are not completely ready for childbirth and if the marriage age is too old the chance of maternal death increases.5,8 From a demographic perspective, this delay can reduce the number of births from the years available for pregnancy, as well as increase understanding for making decisions about fertility choices including determining the number of children.6,7 Not only does it have a positive impact on demographic issues, the study showed that regulating the maturity of the marriage age in the law can protect the rights of women and children and increase their quality of life.9\n\nTables 1, 2 and 3 show that women aged 15-49 who were aged 10-24 at their first marriage have a longer reproductive age range, so they have relatively more children than women who married at the age of 25 and over. The delay in the age of marriage and childbirth leads to a limitation of the biological age of pregnancy.10 Furthermore, this analysis shows that the age of 25 years in women is a good minimum age to be able to give birth to an average of less than 3 children. Research in Uganda indicates that delaying the age of marriage beyond 20 years can change the country’s sexual behavior and fertility rates.11 Muhoza (2022) concluded that the decline in fertility rates was influenced by delays in the age of marriage.12 Other studies suggest that delaying the age of marriage has an impact on delaying obligations in caring for and caring for children, but this factor does not stand alone in determining the number of children you have because the level of education, economic status, and the value of the child also has a large enough contribution on one’s fertility decisions.10,13\n\nRegional characteristics also show a gap in fertility trends and age at first marriage.14 Based on 2017-2019 GPAS/SKAP data, only the Java-Bali region is consistent with a minimum age of first marriage of 25 years for women if they want to have 2 children. Other regions showed varying results in two children and age at first marriage in the 25-29 and 30-34 age groups. The trend of births in Indonesia still show a positive perspective on the relatively small number of children, but there are indications of an increase in births. The report of the 2006-2014 Susenas showed as many as 86% of women aged 15-49 have two children or less because of the success of the happy and prosperous small family program GPAS.15 The results of this study in total, Indonesian women have 2-3 children, with the lowest scores on the islands of Java and Bali and the highest on the island of Maluku. The Java-Bali region has a low average number of children because until now development and family planning programs are still focused in that area.16,17 Other countries in Southeast Asia share similar views regarding small families, for example in Sri Lanka. The study described a fertility preference for only having two or three children, but reported increased fertility for multifactorial reasons.18\n\nThe data in Table 4 shows that after controlling for other factors, women who have a tendency to have more than two children are namely: living in a city, have a high education level, low economy, not working, want to have more than two children, low AFM, and using contraception tools. Several studies, both in Indonesia and in other countries found that women living in rural areas are more likely to have fewer children due to economic limitations.11,19,20 This reason is also consistent with the results of this study which showed that low economic status is in line with low fertility rates as well. However, the results of studies in Europe stated that fertility was lower due to the high rate of delay in pregnancy in urban areas21 and also the results of studies in Nigeria which showed differences with the findings in this study, namely that living in rural areas and the age at first marriage contributed to the high number of children.22,23\n\nThe assumption that those with a high level of education will have a few children was not proven in this study after being controlled by other factors.11 Higher education has a positive correlation with the desire to have two or more children because it is related to “individual agency” in determining their fertility preferences, including religious norms, child values, and ideal preferences for the number of children which are also significant in this study.13 Furthermore, an understanding of fertility encourages women to determine their preference for the number of children and their fertility rights.24\n\nWorking women were positively correlated with having fewer children due to the dual burden of family and work.25 On the other hand, women who do not work have difficulties in making decisions, including decisions about fertility rights, because the “power” lies in the hands of their husbands.26 The odds ratio analysis also showed that a relatively low age at first marriage is directly proportional to a high fertility rate. This finding is also supported by other studies which stated that delaying marriage can reduce fertility rates and protect reproductive rights in women.6,7,10,13 Another interesting finding in this study is that the use of contraceptives has a negative correlation with low fertility rates. This is different from other studies which concluded that contraceptive users have relatively few children compared to those who do not use contraception.11 This study indicates that contraception is only used after having more than two children to limit the number of births, not to regulate the number of births.27\n\nThe limitation of the study is that the data analyzed is still measured using the variable average of children ever born, it has not been measured by the total birth rate (TFR) which has taken into account child mortality. This data uses the aggregate level at the province, while district/city data is needed for programs in urban districts since district/city policies are no longer centralized (decentralization).\n\nThis study has an impact on gender issues, namely Maternal and Child Health. Therefore, proper identification of AFM can provide women’s reproductive rights so that it will reduce the problem of Maternal and Child Mortality. In addition, having less than 2 children can optimize a woman’s role as a mother in caring for her children.\n\n\nConclusion\n\nThe median age at first marriage in Indonesia tends to increase from year to year but is still not as expected. The median age at marriage has broad implications because it provides opportunities for women to prepare for a better future. From the aspect of reproductive health, women will become healthier and feel ready to have children. In addition, the results of the analysis prove that the call for the best age for women to first get married is 25 years, not 21 years which has always been believed. The median age of 25 for first marriage gives women the opportunity to attend higher education, so that they can join the job market.",
"appendix": "Data availability\n\nThe data is available in the institution (National Family Planning Agency in Indonesia/www.bkkbn.go.id). The raw data can be accessed upon request from the institution through email with the data request letter and proposal enclosed (puslitbangkbks@gmail.com).\n\n\nAcknowledgments\n\nWe gratefully acknowledge the National Population and Family Planning Board for providing the opportunity given to utilize the 2017-2019 GPAS/SKAP data and the LPDP (Lembaga Pengelola Dana Pendidikan/Education Fund Management Institution) for financial support.\n\n\nReferences\n\nBadan Pusat Statistik: Indonesia Population Census 2020. Jakarta:2021.\n\nBKKBN: RENCANA STRATEGIS BKKBN 2020-2024. Pertama. Jakarta. Indonesia:Badan Kependudukan dan Keluarga Berencana Nasional;2020.\n\nBKKBN: Panduan Pelaksanaan Sosialisasi Program Pembangunan Keluarga Bersama Mitra Kerja.Vol. 53. 2018; pp. 1689–1699.\n\nCates W: Age and fertility. In: Hospital practice (Office ed).1982; p. 21.\n\nMarphatia AA, Ambale GS, Reid AM: Women’s Marriage Age Matters for Public Health: A Review of the Broader Health and Social Implications in South Asia. Front. Public Health. 2017; 5(269): 1–23.\n\nBaruwa OJ, Amoateng AY, Biney E: Socio-demographic Changes in Age at First Marriage in Malawi: Evidence from Malawi Demographic and Health Survey data, 1992-2016. J. Biosoc. Sci. 2019; 52: 832–845. Publisher Full Text\n\nAktar S, Mondal S, Hossain MA, et al.: Socio-economic Determinants of Age at First Marriage of Rural Women: A Statistical Analysis. Int. J. Eng. Comput. Sci. 2017; 6(12): 22255–22260.\n\nUNFPA: Program of Action: Adopted at the International Conference on Population and Development, Cairo 1994. New York:2004; 1–177.\n\nBatyra E, Pesando LM: Trends in child marriage and new evidence on the selective impact of changes in age-at-marriage laws on early marriage. SSM-Population Heal. 2021; 14(100811): 100811–100812. Publisher Full Text\n\nNitsche N, Hayford SR: Preferences, Partners, and Parenthood: Linking Early Fertility Desires, Marriage Timing, and Achieved Fertility. Demography. 2020; 57: 1975–2001. PubMed Abstract | Publisher Full Text | Free Full Text\n\nAriho P, Kabagenyi A: Age at First Marriage, Age at First Sex, Family Size Preferences, Contraception and Change in Fertility among Women in Uganda: Analysis of the 2006-2016 Period. BMC Womens Health. 2020; 20(8): 1–13. Publisher Full Text\n\nMuhoza DN: Fertility Transition in Rwanda: What Does the Trend in Nuptiality reveal? Genus. 2022; 78(1). Publisher Full Text\n\nUtomo A, McDonald P, Utomo I, et al.: Do Individuals with Higher Education Prefer Smaller Families? Education, Fertility Preference and the Value of Children in Greater Jakarta. Child Indic. Res. 2021; 14(1): 139–161. Publisher Full Text\n\nHertrich V: Trends in Age at Marriage and the Onset of Fertility Transition in Sub-Saharan. Popul. Dev. Rev. 2017; 1–26.\n\nBPS-Statistics Indonesia: Welfare Statistics 2016. Jakarta:2016.\n\nSujarwoto S: Small Family Norms and Family Well-being in Indonesia, 2006-2014. J. Biosoc. Sci. 2017; 49: 96–115.\n\nMahmud A, Ekoriano M, Titisari AS, et al.: Determinants Of Modern Contraceptives Use In Indonesia: A Spatial Analysis.2021; 12(3): 769–777.\n\nDe Silva WI , Goonatilaka WS: Pronatalistic Value of Children and Sri Lanka’s Fertility Rebound. Child Indic. Res. 2021; 14: 607–628. Publisher Full Text\n\nLerch M: Regional Variations in The Rural-Urban Fertility Gradient in The Global South. PLoS One. 2019; 14(7): 1–18. Publisher Full Text\n\nWithers M, Browner CH: The Changing Contexts of Fertility Outcomes: Case Studies from a Remote Village in Bali, Indonesia. Cult. Health Sex. 2012; 14(3): 347–360. PubMed Abstract | Publisher Full Text\n\nRiederer B, Buber-Ennser I: Regional Context and Realization of Fertility Intentions: The Role of The Urban Context. Reg. Stud. 2019; 53(12): 1669–1679. PubMed Abstract | Publisher Full Text | Free Full Text\n\nAlaba OO, Olubusoye OE, Olaomi JO: Spatial patterns and determinants of fertility levels among women of childbearing age in nigeria. South African. Fam. Pract. 2017; 59(4): 143–147. Publisher 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\nSpagnoletti BRM, Bennett LR, Kermode M, et al.: ‘The Final Decision is With The Patient’: Reproductive Modernity and Preferences for Non-Hormonal and Non-Biomedical Contraceptives Among Postpartum Middle Class Women in Yogyakarta, Indonesia. Asian Popul. Stud. 2019; 15: 105–125. Publisher Full Text\n\nOwoo NS, Lambon-Quayefio MP: Does Job Security Affect Fertility and Fertility Intentions in Ghana? Examining the Evidence. J. Fam. Econ. Iss. 2022; 43(1): 86–99. Publisher Full Text\n\nAnnan J, Donald A, Goldstein M, et al.: Taking power: Women’s empowerment and household Well-being in Sub-Saharan Africa. World Dev. 2021; 140: 105292. Publisher Full Text\n\nMaha DMB: Determinan yang Berpengaruh terhadap Pemakaian Alat Kontrasepsi IUD di Puskesmas Kecamatan Kramat Jati Kota Administrasi Jakarta Timur tahun 2013. J. Bid. Ilmu Kesehat. 2018; 11(1): 745–754."
}
|
[
{
"id": "175606",
"date": "14 Jun 2023",
"name": "Hanna Silitonga",
"expertise": [],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis is an important topic, as early marriage remains an important issue in Indonesia. However, the paper could be improved by considering the following comments.\nThe title is not clear. Reading the paper, I think the title may have been “Relationship between Age at First Marriage (AFM) and Children Ever Born among Married Women at the End of Their Reproductive Period in Indonesia”.\nThe paper does not mention its aim explicitly either. Actually, in my interpretation, the paper aims to evaluate whether women who first married at a younger age are related to having higher numbers of children ever born (CEB). The paper makes two categories of CEB, whether it is below two children or otherwise. The threshold 2 is selected by the authors to follow the official state policy that “two” is the ideal number of children per woman. Therefore, it should be noted that different thresholds may provide different results.\nIn the first paragraph of the introduction, the paper should have mentioned that Total Fertility Rate (TFR)s in some provinces have been below replacement rate.\nTo avoid censored information (because the women have not finished their reproductive period), the paper limits the analysis to married women aged 40-49. It should be noted however that this analysis may only represent the relationship between AFM and CEB for older cohorts. Younger cohorts may have different behaviour. Additionally, ever-married women 40-49, excluded from the sample, may have another behaviour.\nThis paper used existing data sets, the SKAP/GPAS, already collected by BKKNN-the National Board of Family Planning and Population, Government of Indonesia every year. The paper used the data for 2017, 2018, and 2019. It has 4 tables and 1 Figure. The first 3 tables discussed the published statistics from the surveys. They showed Children Ever born (CEB) in each age group of Age at First Marriage (AFM) in 2017, 2018, and 2019 by islands in Indonesia. However, it is not clear about the sample used in the tables. Is it married women only? Is it for women aged 15-49, 40-49, or others? The titles of the 3 tables should be changed to make the tables self-explanatory.\n\nInterestingly, the pattern in 2017 (Table 1) is very different from those in 2018 (Table 2) and 2019 (Table 3), especially for Indonesia (total). In 2017, the relationship in Indonesia follows a U-curve; in 2018 and 2019, it has a declining trend. Additionally, the changes from 2017 to 2018 and to 2019 are substantial. For example, in 2017, the CEB for AFM at 45-49 is 2.55 (Table 1). In one year, it declined dramatically to 0.72 in 2018 (Table 2). Is there anything wrong with the data or tables? Moreover, the paper does not have AFM at 45-49 in 2019 (Table 3). Therefore, the paper needs to address this issue, including why there is no column for AFR at 45-49 in 2019 (Table 3).\nIt should also be mentioned that, in some regions, in each of the three years, the relationships are also fluctuating. It is then difficult to conclude a negative relationship between AFM and CEB from Tables 1, 2, and 3. The paper should have addressed this issue more.\nFigure 1 shows that TFR in 2019 is 2.45, very different from the result of 2020 population census, which is much lower, only 2.18. The result of the 2020 population census used an indirect method, the own children method, referring to the past several years. The TFRs in SKAP/ GPAS used a direct method, giving estimates in the same year. There are also some other estimates of TFR, such as those produced by Statistics-Indonesia. The paper should examine other estimates of TFR as fertility is one of two main variables in the paper with the other one in AFM. In addition to other estimates of TFR, the paper could also analyse alternative estimates of AFM.\nThe paper should have explained what we can learn about the relationship between AFM and CEB from Tables 1,2, and 3.\nThe paper should have done a literature study on AFM in Indonesia and should have compared the results of this paper with previous studies in Indonesia.\nIt should be noted that the sample of Table 4 is limited to married women aged 40-49. It is not clear whether the sample is different from those in Tables 1, 2, and 3. The TFR also refers to a “current” situation rather than past situation as in CEB. Therefore, a comparison of results in Tables 1, 2, and 3 on one hand and Table 4, on the other hand, should be done with caution.\nThe paper has detailed how the surveys were conducted by BKKBN. However, the paper should explain more about the method of analysis (including the statistical tool) and sample of data used in the analysis.\nTable 4 utilized the individual data sets. The paper runs logistic regression for each year: 2017, 2018, and 2019. The dependent variable is a dummy, whether the CEB is <2 or otherwise. However, there is a typing error in the first column of Table 4. One of the independent variables was “number of children”. It should have been “ideal/desired number of children”. Since it was asked at the end of the reproductive period, the authors may explain the possibility that the answer to the ideal number has been adjusted to the actual number.\nFrom Table 4, the paper concluded that AFM had the dominant contribution to CEB. It should explain how the conclusion is made. If the threshold is not 2, but a different number, will the result be different? How about if the paper uses a continuous number of CEB – 0,1, 2,3, etc? The paper should have addressed more on how the paper is related to the state policy of 2 being the ideal number of children.\nBefore the discussion, the paper wrote “For women aged 15-49, more than 2 children is the ideal number desired, namely in 2018 (AOR: 6.55 99% CI [5.97-7.19] and in 2019 (AOR 6.58 99% [6.11-7.08])”. Is “15-49” correct? Or, is it “40-49”? It is not clear what is meant by “more than 2 children is the ideal number desired”\nUnder the discussion, the paper mentions rights and quality of life. Does the paper have the measurements of these two variables? Are they included in the statistical analysis?\nUnder discussion, the paper should have integrated the results of Tables 1, 2, and 3 and Figure 1 with those from Table 4.\nThe paper may have missed two important proximate determinants of fertility – fecundity (often indicated with amenorrhea) and breastfeeding. It should have considered these missing variables in the discussion.\nThe paper used contraceptive use without differentiating between modern and traditional ones, though the differentiation may have an important impact on CEB. The paper should have addressed this issue.\nThe paper concludes that the best median women’s age at first marriage should be 25, not 21, to have CEB under 2. The paper should explain more about how this conclusion is made. As the sample is old reproductive-age women, the conclusion may not be applicable to younger women.\nFinally, the quality of the paper could be improved by having copy editing, which could include English editing. The authors could also improve the flow of discussion, to minimize the possibility of misunderstanding the paper.\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Partly\n\nAre sufficient details of methods and analysis provided to allow replication by others? Partly\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nPartly\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Partly",
"responses": []
}
] | 1
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https://f1000research.com/articles/12-35
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https://f1000research.com/articles/11-269/v1
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03 Mar 22
|
{
"type": "Research Article",
"title": "Dose response relationship between D-dimer level and mortality in critically ill COVID-19 patients: a retrospective observational study",
"authors": [
"Dita Aditianingsih",
"Ratna Farida Soenarto",
"Artheta Mutiara Puiantana",
"Raymond Pranata",
"Michael Anthonius Lim",
"Putu Angga Risky Raharja",
"Ponco Birowo",
"Markus Meyer",
"Ratna Farida Soenarto",
"Artheta Mutiara Puiantana",
"Raymond Pranata",
"Michael Anthonius Lim",
"Putu Angga Risky Raharja",
"Ponco Birowo",
"Markus Meyer"
],
"abstract": "Background: Coronavirus disease 2019 (COVID-19) is a global pandemic. Coagulopathy is one of the most common complications characterized by increased D-dimer level. We aimed to investigate the dose-response relationship between elevated D-dimer level and mortality in critically ill COVID-19 patients. Methods: This was a retrospective observational study in 259 critically ill COVID-19 patients requiring intensive care unit admission between March and December 2020. We compared the mortality rate between patients with and without elevated D-dimer. Receiver operating characteristic (ROC) curve analysis, Fagan’s nomogram, and dose-response relationship were performed to determine the association between D-dimer level and mortality. Results: Overall mortality rate was 40.9% (106 patients). Median D-dimer level was higher in non-survivor group (10,170 ng/mL vs 4,050 ng/mL, p=0.028). The association remained significant after multivariate logistic regression analysis (p=0.046). The optimal cut-off for D-dimer level to predict mortality from ROC curve analysis was 9,020 ng/mL (OR (odds ratio) 3.73 [95% CI (confidence interval) 1.91 – 7.28], p<0.001). D-dimer level >9,020 ng/mL confers 67% posterior probability of mortality and D-dimer level <9,020 ng/mL had 35% probability of mortality. Conclusions: There was a non-linear dose-response relationship between D-dimer level and mortality with Pnonlinearity of 0.004. D-dimer level was associated with mortality in critically ill COVID-19 patients in the non-linear dose-response relationship.",
"keywords": [
"COVID-19",
"critically ill",
"D-dimer",
"dose-response relationship",
"mortality"
],
"content": "Introduction\n\nCurrently, the coronavirus disease 2019 (COVID-19) is one of the most common diseases affecting society globally, causing a sizeable number of deaths as of 2022.1 Although the majority of patients had only mild-moderate clinical manifestations, a small but significant proportion developed life-threatening complications, for example, multiple organ failure.2–4 This is especially true in patients with comorbidities, such as diabetes mellitus (DM), chronic kidney disease (CKD), and cerebrovascular and cardiovascular diseases, that are associated with increased severity and mortality from COVID-19.5–11 The cases of COVID-19 remain high and threaten to overwhelm the healthcare system, therefore risk stratification is required for prudent allocation of resources. Several biomarkers have been evaluated or repurposed to attain this objective.12–14\n\nThe importance of simple laboratory values, such as complete blood count, coagulation parameters, and inflammatory biomarkers, cannot be underestimated during the ongoing COVID-19 pandemic.15–18 These values can help predict the severity of the disease course and often indicate the need for intensive care unit (ICU) admission or mechanical ventilation. Coagulopathy is among the most frequently found complications, which is characterized by the elevation of D-dimer level and changes in fibrinogen levels and platelet counts.19,20 Although increased D-dimer levels have been consistently observed in severely and critically ill patients, the optimal cut-off level and prognostic values remain unknown.21 This study aimed to investigate the dose-response relationship between elevated D-dimer level and mortality in critically ill COVID-19 patients.\n\n\nMethods\n\nThis study was conducted in accordance with the ethical standards of the Helsinki Declaration. The Ethics Committee of the Faculty of Medicine, Universitas Indonesia (date 15.06.20, No. 0593/UN2.F1/ETIK/PPM.00.02/2020) approved the study protocol. Informed consent was not obtained because the study was retrospective observational in nature.\n\nThis study was a retrospective observational study in Cipto Mangunkusumo Hospital and Universitas Indonesia Hospital, Indonesia. There were 261 critically ill COVID-19 patients requiring intensive care unit (ICU) admission between March and December 2020. COVID-19 diagnosis was based on a reverse transcriptase-polymerase chain reaction (RT-PCR) examination. We did consecutive enrollment to address any potential sources of bias. Data on the patients’ baseline clinical and laboratory characteristics were collected in a list form from the medical records in December 2020 and then analyzed using IBM SPSS Statistics version 25. We used a multivariate analysis to adjust the possible effect of confounders.\n\nThe outcome of this study was mortality, defined as clinically validated death/non-survivor. The outcome was ascertained from the medical record and confirmed by the death certificate. An independent investigator of the data collection process and patient care performed the statistical analysis. We compared the mortality rate between the patients with elevated D-dimer level and those without.\n\nWe performed the statistical analysis using SPSS 25.0 (Armonk, US) and STATA 14.0 (College Station, TX, US). We tested continuous data for normal distribution; t-test was used for normally distributed data, and Mann-Whitney test was used for abnormally distributed data. Chi-square test or Fischer-Exact test was performed for categorical variables. Normally distributed continuous data were presented as mean and standard deviation (SD); while abnormally distributed continuous data were reported as the median and interquartile range (IQR). Bivariate analysis was performed to evaluate variables. Continuous variables were compared using either independent T or Mann-Whitney U test. Categorical variables were tested by using chi-square test. Significant variables (P<0.05) were included in the logistic regression analysis. ROC curve analysis was performed to determine the optimal cut-off points for the D-dimer level. The sensitivity, specificity, positive likelihood ratio (LR+), negative likelihood ratio (LR-), and area under the curve (AUC) were calculated. Fagan’s nomogram was plotted to determine the posterior probability of mortality for the cut-off points determined by ROC curve analysis. The dose-response relationship between D-dimer level and mortality in the patients was explored and reported in the form of a restricted cubic spline.\n\n\nResults\n\nThis study enrolled 261 critically ill COVID-19 patients between March 2020 and December 2020 in this study. Two patients were excluded due to missing data, so 259 patients who met the inclusion criteria were included in the analysis. The mortality rate was 40.9% (106 patients). Demographic characteristics, preoperative laboratory parameters, and comorbidities of the patients were presented in Table 1. Patients in the non-survivor group were older than the survivor group. The mean ages of the non-survivor group and survivor group are 56.04 years and 46.73 years, respectively (p<0.001). There was no significant difference in gender distribution between groups. Non-survivors were more likely to have higher D-dimer levels, leukocyte levels, C-reactive protein levels, and lower thrombocyte levels. Comorbidities such as hypertension, coronary artery disease, and chronic kidney disease were more frequently found in the non-survivor group.\n\nMedian D-dimer level was higher in the non-survivor group (10,170 ng/mL vs 4,050 ng/mL, p=0.028). This association remained significant after logistic regression multivariate analysis (p=0.046), as shown in Table 2.\n\nThe optimal cut-off level for D-dimer to predict mortality in critically ill COVID-19 patients was determined to be 9,020 ng/mL (OR 3.73 [95% CI 1.91 – 7.28], p<0.001) through ROC curve analysis. It was associated with 52% sensitivity, 78% specificity, LR+2.31, and LR- 0.62. The AUC was 0.657 (95% CI 0.573-0.740), p=0.001 [Figure 1]. In our patients, a D-dimer level of >9,020 ng/mL gave 67% posterior probability of mortality, and a D-dimer level of <9,020 ng/mL had 35% probability of mortality [Figure 2]. There was a non-linear dose-response relationship between D-dimer level and mortality with Pnonlinearity=0.004 [Figure 3].\n\n\nDiscussion\n\nThis study showed that D-dimer level has a non-linear dose-relationship with mortality in critically ill COVID-19 patients. The optimal D-dimer level cut-off point was 9,020 ng/mL. An elevation beyond the cut-off point confers to 67% posterior probability of mortality, and D-dimer level of <9,020 ng/mL had 35% probability of mortality.\n\nD-dimer is a fragment produced when plasmin cleaves fibrin during clot breakdown. In clinical practice, D-dimer assays are frequently used to exclude a diagnosis of deep vein thrombosis (DVT) and pulmonary embolism (PE). Abnormal coagulation function, specifically blood clotting, is often indicated by an elevation in D-dimer level, reflecting a severe viral infection. Elevated D-dimer level is associated with COVID-19 progression as well as a greater death rate from community-acquired pneumonia (CAP).17,22 For this reason, testing D-dimer level on admission could be useful to stratify patients with COVID-19 early and to determine the treatment plan. A high on-admission D-dimer level might suggest a prothrombotic state, increased fibrinolysis, bleeding, and thrombotic events, and indicate cytokine storm, tissue damage, or impending sepsis.17,23\n\nElevated D-dimer level has been shown to be associated with mortality in COVID-19 patients.17 This study showed that D-dimer level can be used for prognostication in critically ill COVID-19 patients and also showed the non-linear dose-response relationship of D-dimer level and mortality. Elevation of inflammatory cytokines and biomarkers such as interleukin (IL)-2, IL-6, IL-7, granulocyte-colony stimulating factor, macrophage inflammatory protein 1-α, tumor necrosis factor-α, C-reactive protein (CRP), ferritin, procalcitonin (PCT), and D-dimer level were found in the systemic hyperinflammation phase.17,24 Excessive hyperinflammation consequently leads to multi-organ failure24–26; therefore D-dimer level may reflect the severity of inflammation. Additionally, SARS-CoV-2 can induce hypercoagulability,27,28 causing thrombosis in cardiovascular systems as well as myocardial injuries.29 Coagulopathy and cardiac injury results in higher mortality in patients with COVID-19,3,26,30,31 which may explain the relationship between D-dimer level and mortality in COVID-19 patients.\n\nThere is a crosstalk between blood coagulation and inflammation. The release of pro-inflammatory cytokines results in the upregulation of tissue factor (TF) expression on the endothelial cells and monocytes surfaces, which further promotes procoagulant activity.32 Moreover, hypoxia in COVID-19, CAP, or other illnesses may stimulate thrombosis by upregulating the hypoxia-inducible transcription factors which modulate the expression of various coagulation and fibrinolytic factors such as TF, tissue factor pathway inhibitor, and plasminogen activator inhibitor-1 (PAI-1).33 In addition, neutrophil extracellular traps, in which neutrophil infiltrates lung capillaries and cause fibrin deposition and acute inflammation, may exacerbate COVID-19 progression by causing organ damage and promoting thrombosis.34\n\nInternational Society of Thrombosis and Haemostasis (ISTH) guideline states that an elevated D-dimer levels indicates increased thrombin production. COVID-19 patients with markedly elevated D-dimer level may require hospitalization despite the severity of clinical presentation.17,35 Prophylactic anticoagulant is recommended in hospitalized patients with COVID-19 in the absence of contraindication.\n\nA limitation of this study was the small sample size of patients from two centers that needed further external validation to support the clinical practice. This research began at the beginning of the pandemic, hence there were adjustments to the system for recording and storing medical records for COVID-19 patients. Secondary data was taken from the medical records, which had a risk of selection, recall, or misclassification bias. This study is a cross-sectional descriptive study, which was influenced by several data biases such as data availability, different follow-up examination, and different given therapies because several patients underwent another different study as this study was held.\n\n\nConclusion\n\nD-dimer level was associated with mortality in critically ill COVID-19 patients in the non-linear dose-response relationship.\n\n\nData availability\n\nfigshare: Data Set (D-Dimer).xlsx. https://doi.org/10.6084/m9.figshare.17125520.v5\n\nThis project contains the following files:\n\n- Data Set (D-Dimer) – Outcome.xlsx (Outcome of the subjects whether died or stepped down from ICU)\n\n- Data Set (D-Dimer) – Characteristics.xlsx (Characteristics of the subjects, such as gender, age, body weight, comorbidities, and lab result)\n\n- Data Set (D-Dimer) – Lab.xlsx (Lab result from each subject)\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).",
"appendix": "References\n\nWorld Health Organization: Weekly Epidemiological Update - 2 March 2021. 2021. Reference Source\n\nLim MA, et al.: Multiorgan Failure With Emphasis on Acute Kidney Injury and Severity of COVID-19: Systematic Review and Meta-Analysis. Can. J. Kidney Health Dis. 2020; 7: 2054358120938573. PubMed Abstract | Publisher Full Text\n\nPranata R, Huang I, Raharjo SB: Incidence and impact of cardiac arrhythmias in coronavirus disease 2019 (COVID-19): A systematic review and meta-analysis. Indian Pacing Electrophysiol. J. 2020; 20: 193–198. PubMed Abstract | Publisher Full Text\n\nPranata R, Huang I, Lim MA, et al.: Delirium and Mortality in Coronavirus Disease 2019 (COVID-19) – A Systematic Review and Meta-analysis. Arch. Gerontol. Geriatr. 2021; 95: 104388. PubMed Abstract | Publisher Full Text\n\nPranata R, Huang I, Lim MA, et al.: Impact of cerebrovascular and cardiovascular diseases on mortality and severity of COVID-19–systematic review, meta-analysis, and meta-regression. J. Stroke Cerebrovasc. Dis. 2020; 29: 104949. PubMed Abstract | Publisher Full Text\n\nPranata R, et al.: The Association Between Chronic Kidney Disease and New Onset Renal Replacement Therapy on the Outcome of COVID-19 Patients: A Meta-analysis. Clin Med Insights Circ Respir Pulm Med. 2020; 14: 1179548420959165. PubMed Abstract | Publisher Full Text\n\nPranata R, Lim MA, Huang I, et al.: Hypertension is associated with increased mortality and severity of disease in COVID-19 pneumonia: A systematic review, meta-analysis and meta-regression. J. Renin-Angiotensin-Aldosterone Syst. 2020; 21: 1470320320926899. PubMed Abstract | Publisher Full Text\n\nPranata R, et al.: Body mass index and outcome in patients with COVID-19: A dose–response meta-analysis. Diabetes Metab. 2021; 47: 101178. PubMed Abstract | Publisher Full Text\n\nHuang I, Lim MA, Pranata R: Diabetes mellitus is associated with increased mortality and severity of disease in COVID-19 pneumonia – A systematic review, meta-analysis, and meta-regression: Diabetes and COVID-19. Diabetes Metab. Syndr. Clin. Res. Rev. 2020; 14: 395–403. PubMed Abstract | Publisher Full Text\n\nPranata R, et al.: Effect of chronic obstructive pulmonary disease and smoking on the outcome of COVID-19. Int. J. Tuberc. Lung Dis. 2020; 24: 838–843. PubMed Abstract | Publisher Full Text\n\nKuswardhani TRA, Henrina J, Pranata R, et al.: Charlson comorbidity index and a composite of poor outcomes in COVID-19 patients: A systematic review and meta-analysis. Diabetes Metab. Syndr. Clin. Res. Rev. 2020; 14: 2103–2109. PubMed Abstract | Publisher Full Text\n\nPranata R, et al.: Fibrosis-4 index and mortality in coronavirus disease 2019. Eur. J. Gastroenterol. Hepatol. 2021; 33: e368–e374. PubMed Abstract | Publisher Full Text Reference Source\n\nMartha JW, Wibowo A, Pranata R: Prognostic value of elevated lactate dehydrogenase in patients with COVID-19: A systematic review and meta-analysis. Postgrad. Med. J. 2021; 0: 1–6. PubMed Abstract | Publisher Full Text\n\nAkbar MR, et al.: The prognostic value of elevated creatine kinase to predict poor outcome in patients with COVID-19 - A systematic review and meta-analysis: Creatinine Kinase in COVID-19. Diabetes Metab. Syndr. Clin. Res. Rev. 2021; 15: 529–534. PubMed Abstract | Publisher Full Text\n\nHuang I, Pranata R: Lymphopenia in severe coronavirus disease-2019 (COVID-19): Systematic review and meta-analysis. J. Intensive Care. 2020; 8: 36. PubMed Abstract | Publisher Full Text\n\nPranata R, et al.: Thrombocytopenia as a prognostic marker in COVID-19 patients: Diagnostic test accuracy meta-analysis. Epidemiol. Infect. 2021; 149: e40. PubMed Abstract | Publisher Full Text\n\nHuang I, Pranata R, Lim MA, et al.: C-reactive protein, procalcitonin, D-dimer, and ferritin in severe coronavirus disease-2019: a meta-analysis. Ther. Adv. Respir. Dis. 2020; 14: 175346662093717–175346662093714. PubMed Abstract | Publisher Full Text\n\nYonas E, et al.: Elevated interleukin levels are associated with higher severity and mortality in COVID 19 – A systematic review, meta-analysis, and meta-regression. Diabetes Metab. Syndr. Clin. Res. Rev. 2020; 14: 2219–2230. PubMed Abstract | Publisher Full Text\n\nGrobler C, et al.: Covid-19: The rollercoaster of fibrin (ogen), d-dimer, von willebrand factor, p-selectin and their interactions with endothelial cells, platelets and erythrocytes. Int. J. Mol. Sci. 2020; 21: 1–25. PubMed Abstract | Publisher Full Text\n\nZhang X, Yang X, Jiao H, et al.: Coagulopathy in patients with COVID-19: a systematic review and meta-analysis. Aging. 2020; 12: 24535–24551. PubMed Abstract | Publisher Full Text\n\nAl-Ani F, Chehade S, Lazo-Langner A: Thrombosis risk associated with COVID-19 infection. A scoping review. Thromb. Res. 2020; 192: 152–160. PubMed Abstract | Publisher Full Text\n\nYu HH, Qin C, Chen M, et al.: D-dimer level is associated with the severity of COVID-19. Thromb. Res. 2020; 195: 219–225. PubMed Abstract | Publisher Full Text\n\nSakka M, et al.: Association between D-Dimer levels and mortality in patients with coronavirus disease 2019 (COVID-19): a systematic review and pooled analysis. J. Med. Vasc. 2020; 45: 268–274. Publisher Full Text\n\nSiddiqi HK, Mehra MR: COVID-19 Illness in Native and Immunosuppressed States: A Clinical-Therapeutic Staging Proposal. J. Heart Lung Transplant. 2020; 39: 405–407. PubMed Abstract | Publisher Full Text\n\nZhang W, et al.: The use of anti-inflammatory drugs in the treatment of people with severe coronavirus disease 2019 (COVID-19): The experience of clinical immunologists from China. Clin. Immunol. 2020; 214: 108393. PubMed Abstract | Publisher Full Text\n\nLim MA, et al.: Multiorgan Failure With Emphasis on Acute Kidney Injury and Severity of COVID-19: Systematic Review and Meta-Analysis. Can. J. Kidney Health Dis. 2020; 7: 2054358120938573. PubMed Abstract | Publisher Full Text\n\nLevi M, van der Poll T : Coagulation and sepsis. Thromb. Res. 2017; 149: 38–44. Publisher Full Text\n\nLin L, Lu L, Cao W, et al.: Hypothesis for potential pathogenesis of SARS-CoV-2 infection--a review of immune changes in patients with viral pneumonia. Emerg. Microbes Infect. 2020; 9: 727–732. PubMed Abstract | Publisher Full Text\n\nLue W, et al.: Clinical Pathology of Critical Patient with Novel Coronavirus Pneumonia (COVID-19).2020. Reference Source\n\nWibowo A, Pranata R, Akbar MR, et al.: Prognostic performance of troponin in COVID-19: A diagnostic meta-analysis and meta-regression. Int. J. Infect. Dis. 2021; 105: 312–318. PubMed Abstract | Publisher Full Text\n\nWibowo A, et al.: Left and right ventricular longitudinal strains are associated with poor outcome in COVID-19: a systematic review and meta-analysis. J. Intensive Care. 2021; 9: 9. PubMed Abstract | Publisher Full Text\n\nOkamoto T, Suzuki K: The Role of Gap Junction-Mediated Endothelial Cell-Cell Interaction in the Crosstalk between Inflammation and Blood Coagulation. Int. J. Mol. Sci. 2017; 18: 2254. PubMed Abstract | Publisher Full Text\n\nGupta N, Zhao YY, Evans CE: The stimulation of thrombosis by hypoxia. Thromb. Res. 2019; 181: 77–83. PubMed Abstract | Publisher Full Text\n\nBarnes BJ, et al.: Targeting potential drivers of COVID-19: Neutrophil extracellular traps. J. Exp. Med. 2020; 217: e20200652. PubMed Abstract | Publisher Full Text\n\nThachil J, et al.: ISTH interim guidance on recognition and management of coagulopathy in COVID-19. J. Thromb. Haemost. 2020; 18: 1023–1026. PubMed Abstract | Publisher Full Text"
}
|
[
{
"id": "126112",
"date": "14 Mar 2022",
"name": "Martin Westphal",
"expertise": [
"Reviewer Expertise Anesthesiology and intensive care"
],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 and well conducted retrospective observational study in COVID-19 patients addressing a clinically relevant topic, i.e. the link between D-dimer levels and mortality.\nPlease be so kind and clarify the discrepancy in patient number reported in the abstract (259) and the study design (261), which just becomes obvious in the results section. Please clarify also, which data were missing, justifying exclusion. Were these exclusion criteria mentioned a priori in the study design/CRF?\nThe rather low sensitivity of the D-dimer levels to predict mortality represents a limitation that should be acknowledged and discussed. Are there any means to increase sensitivity, e.g. by combining 2 or more variables?\n\nIs the work 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": "147444",
"date": "07 Sep 2022",
"name": "Reza Zolfaghari Emameh",
"expertise": [
"Reviewer Expertise Biotechnology",
"Bioinformatics",
"Carbonic anhydrase",
"SARS-CoV-2",
"COVID-19"
],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address 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 “Dose response relationship between D-dimer level and mortality in critically ill COVID-19 patients: a retrospective observational study” was reviewed carefully. The language is acceptable and the subject has great importance, although it is not the first report on the association of D-dimer high concentration with high mortality rate. Please see the following publication: https://www.scielo.br/j/clin/a/w3HnMz3KjhCfSJWVhfKMjRS/?format=pdf&lang=en.\nPlease state briefly about the literature on other factors having an association with high mortality rate or prepare a brief table with proper citations in the “Introduction”, such as PMID: 325723341, PMID: 327540042, PMID: 334078003, PMID: 330832874, and PMID: 360439225.\n\nTable 1: Why the diabetes survivor group has a higher percentage than the non-survivor group? I think diabetes patients are in the high-risk group.\n\nTable 1: Why the thrombocyte percentage is higher in the survivor group than the non-survivor group? High thrombocyte count is one of the main reasons for VTE in COVID-19.\nTherefore, after performing the modifications and due to the decision of the respected editor, this manuscript can be indexed.\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? 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": "8915",
"date": "10 Jan 2023",
"name": "Dita Aditianingsih",
"role": "Author Response",
"response": "First, thank you for the review. We will add the article as you suggested. We agree with the reviewer that diabetic patients have a higher risk. Although the diabetes survivor was higher, there was no significant difference in our patient for diabetes comorbidities (P =0.340), and it could be caused by the average younger age in the survivor group compared to the non-survivor group. Thrombocytopenia is recognized as a negative prognostic factor in COVID-19 patients. Thrombocytopenia is detected in 5–41.7% of COVID-19 patients (the incidence varies according to disease severity) and it is typically mild. We briefly put it in the Introduction with some literature backgrounds."
}
]
}
] | 1
|
https://f1000research.com/articles/11-269
|
https://f1000research.com/articles/11-802/v1
|
18 Jul 22
|
{
"type": "Research Article",
"title": "Genomic prediction in plants: opportunities for ensemble machine learning based approaches",
"authors": [
"Muhammad Farooq",
"Aalt D.J. van Dijk",
"Harm Nijveen",
"Shahid Mansoor",
"Dick de Ridder",
"Muhammad Farooq",
"Aalt D.J. van Dijk",
"Harm Nijveen",
"Shahid Mansoor"
],
"abstract": "Background: Many studies have demonstrated the utility of machine learning (ML) methods for genomic prediction (GP) of various plant traits, but a clear rationale for choosing ML over conventionally used, often simpler parametric methods, is still lacking. Predictive performance of GP models might depend on a plethora of factors including sample size, number of markers, population structure and genetic architecture. Methods: Here, we investigate which problem and dataset characteristics are related to good performance of ML methods for genomic prediction. We compare the predictive performance of two frequently used ensemble ML methods (Random Forest and Extreme Gradient Boosting) with parametric methods including genomic best linear unbiased prediction (GBLUP), reproducing kernel Hilbert space regression (RKHS), BayesA and BayesB. To explore problem characteristics, we use simulated and real plant traits under different genetic complexity levels determined by the number of Quantitative Trait Loci (QTLs), heritability (h2 and h2e), population structure and linkage disequilibrium between causal nucleotides and other SNPs. Results: Decision tree based ensemble ML methods are a better choice for nonlinear phenotypes and are comparable to Bayesian methods for linear phenotypes in the case of large effect Quantitative Trait Nucleotides (QTNs). Furthermore, we find that ML methods are susceptible to confounding due to population structure but less sensitive to low linkage disequilibrium than linear parametric methods.\nConclusions: Overall, this provides insights into the role of ML in GP as well as guidelines for practitioners.",
"keywords": [
"Genomic Prediction",
"Machine Learning",
"Genomic Selection",
"Linear Mixed Models"
],
"content": "Abbreviations\n\nANN: artificial neural network\n\nBLUPs: Best Linear Unbiased Predictions\n\nGBLUP: Genomic Best Linear Unbiased Prediction\n\nGP: Genomic Prediction\n\nMLP: Multilayer Perceptron\n\nQTL: Quantitative Trait Loci\n\nQTN: Quantitative Trait Nucleotide\n\nRF: Random Forest\n\nRKHS: Reproducing Kernel Hilbert Spacing\n\nSNP: single nucleotide polymorphism\n\nSVM: Support Vector Machine\n\nSVR: Support Vector Regression\n\nXGBoost: Extreme Gradient Boosting\n\n\nIntroduction\n\nThe phenotype of an individual is based on its genetic makeup, the environment and the interplay between them. In plant and animal breeding, the genomic prediction (GP) model, using a genome-wide set of markers, is an integral component of the genomic selection-based approach.1 A GP model is constructed on a reference population for which both genotypes and corresponding phenotypes are known, mostly employing a cross-validation strategy, and applied to related populations with only genotypes known. The total genomic value, estimated from the GP model, is used as a pseudo-phenotype to select the best parents for the next generation(s). In general, phenotypes differ from each other in terms of their genetic complexity, ranging from simple/monogenic to complex/polygenic. These differences impact the potential performance of GP. Complex traits are predominantly governed by a combination of additive and non-additive (e.g. dominant/recessive, epistatic etc.) allele effects, which makes GP challenging for these traits.2 The genetic architecture of complex traits is characterized by moderate to large numbers of Quantitative Trait Loci (QTLs) with small to medium effect sizes and no or few large effect QTLs.3 Moreover, the ratio of additive to non-additive genetic variance may differ even for closely related traits. Besides the actual genetic variance level, its distribution over the genome is also a determinant of the trait architecture.4 Next to genetic architecture, population structure plays a role as well (Figure 1): prediction accuracies are influenced by inconsistent relatedness among samples due to ancestral allele frequency imbalance among sub-populations (population structure) or cryptic structures, e.g. familial relationships; linkage disequilibrium (LD) structure, due to inbreeding or selection pressure; varying relatedness between training and test populations, e.g. over the course of a breeding cycle; and sizes of reference and effective populations.5\n\nFactors affecting genomic prediction performance, often measured as correlation between true phenotype values and those predicted by a model.\n\nTechnological advances and statistical frameworks used bring new challenges (Figure 1). Genotyping and/or phenotyping technologies can now generate millions of markers and thousands of phenotypic measurements, e.g. in time series, increasing the dimensionality of the prediction problem. For example, using a high-density SNP array (or imputing SNPs based on a low-density array) increases the likelihood of getting many markers in LD with the true QTL (high SNP-QTL LD). It can increase total explained variance,6 but may induce multicollinearity among SNPs. Consequently, SNP selection prior to predictive modelling has been reported to provide superior performance compared to simply using a dense marker set.7 In contrast, low-density genotyping can miss important SNPs in LD with, or weakly linked to, the QTLs, leading to inferior prediction performance.8\n\nStatistical genetics approaches have traditionally focused on formulating phenotype prediction as a parametric regression of one or more phenotypes on genomic markers, treating non-genetic effects as fixed or random in a linear equation. The resulting GP models are biologically interpretable but might yield poor performance for complex phenotypes, as linear regression fails to capture the more complex relations.9 This approach also requires proper translation of prior knowledge on the genetics underlying phenotypes into parametric distributions. Although statistical distributions can help describe genetic architecture, devising a specific distribution for each phenotype is impractical. Therefore, many variations of linear regression were proposed by relaxing statistical assumptions; the main differences lie in their estimation framework and prior assumptions on the random effects (for an overview, see ‘Models’). Alternatively, machine learning (ML) offers a more general set of non-parametric methods that can model phenotypes as (non) linear combinations of genotypes. Moreover, these methods can jointly model the problem, e.g. strong learners can be stacked10 or weak learners can be combined in an ensemble. Examples include Support Vector Machines (SVMs), (ensembles of) decision trees and artificial neural networks (ANNs). No statistical assumptions are required in advance; therefore, these methods should be able to pick up more complex genetic signals that are missed by linear models. The downside is the large amount of data required for learning these models from the data.\n\nThe performance of ML methods in GP problems has previously been compared using simulated and real phenotypes. Some were found to perform better under non-additive allelic activity11,12; however, a clear link between simulated and real phenotypes is often missing, or only a specific breeding population structure is considered. For example, Barbosa et al.13 compared the performance of ML and statistical methods in a simulated F2 population of 1,000 individuals and 2,010 SNPs using 26 simulated phenotypes. They varied the heritability and number of QTLs and included dominant and epistatic effects. They observed that ML methods performed better at low QTL numbers and hypothesized that a reason for this is that with fewer controlling genes, nonlinear epistatic interactions are more important. But it is still unclear if this is a general conclusion towards a population with different characteristics e.g. natural populations. Moreover, there are conflicting reports on performance of ML.11,14 For example, ANNs have been reported to perform worse in some applications and are comparable to competing methods in others.12,15 Ensemble decision tree methods, combining the output of a large number of simple predictors, have proven better for some traits but not for others.16–18 Gradient boosting showed improved performances for many real traits19,20https://paperpile.com/c/ZyQHHy/b9hH+ha6M but was inferior to random forests on simulated datasets.18 Furthermore, the impact of population structure and low SNP-QTL LD on the performance of ML methods is still unclear.\n\nIn this paper, we investigate which GP characteristics (genetic architecture, population properties and genotype/phenotype measurement technology) a priori point to a better performance for either traditional statistical approaches or ML-based methods. We compare GP performance of two ensemble methods, Random Forests (RF) and Extreme Gradient Boosting (XGBoost), to that of linear mixed models, GBLUP, BayesA, BayesB and RKHS regression with averaged multi-Gaussian kernels. We focus on typical applications in plant breeding to explore various GP characteristics, including the ratio of the total number of markers to the number of samples (p/n), genetic complexity, QTN effect sizes and distributions, linear (additive) vs. nonlinear (epistatic) heritabilities, sparse vs. dense genotyping and population structure.\n\n\nMethods\n\nSimulations\n\nIn a first experiment, artificial genotypes were simulated, in combination with associated phenotype values. Genotype data was simulated for a diploid population with a minor allele frequency of 0.4, using a binomial distribution, where each allele was the outcome of a binomial trial. The genotype dataset was coded as {0=AA, 1=Aa, 2=aa}. To explore GP characteristics (Figure 1), different levels of genetic complexity and dimensionality, defined as the ratio of total number of SNPs to the sample size (c = p/n), were simulated. For the high dimensionality scenarios, sample size was fixed at n = 500, because reference populations of this size are feasible for genotyping and phenotyping in genomic selection studies. Using values of c = {2, 10, 20, 40, 120}, the number of SNPs varied up to p = 60,000 (120*500). Similarly, for the low dimensionality scenarios, the number of SNPs was fixed at p = 500 and sample size was varied up to n = 3,000 to arrive at c = {1, 1/2, 1/4, 1/6}. Subsequently, Quantitative Trait Nucleotides (QTNs) were randomly selected from these simulated SNP sets to generate phenotypes. We selected either 5, 50, 100, p/2 or p QTNs, corresponding to a range of low to high genetic complexity, coupled with a narrow-sense heritability ranging from 0.1 to 0.7. A phenotype with a high number of QTNs and low heritability is more complex than one with few QTNs and higher heritability.\n\nPhenotype datasets were generated using the simplePHENOTYPES v1.3.0 (RRID:SCR_022523) R package.21 Linear polygenic phenotypes were simulated using additive modes of allele effects, as follows:\n\nHere, βi describes the effect size of the ith QTN, where QTNi is a vector containing the allele dosages for the ith QTN for all samples. The residuals (ε) were sampled from a normal distribution N(0, √(1-h2)). The narrow-sense heritability (h2) determines the effect sizes: each QTN is assigned an effect size of βi = h /n, referred to as ‘simulations with equal/uniform effects’ in the text. Smaller effect sizes can be generated by increasing the number of QTNs, thereby simulating higher genetic complexity. To further explore genetic complexity, we used equation (1) to generate another set of phenotypes where the first QTN is assigned a larger effect than others, 2h, and the remainder still h/ (n-1). Accordingly, another set of simulations was generated by sampling effect sizes from N(0, √h2).\n\nFor nonlinear phenotypes, broad-sense heritability was set at most to 0.8, so the distribution of residuals is N(0,√0.2). We considered only epistasis to induce nonlinearity, ignoring other factors such as dominance. Adding an additional term for epistasis to equation (1) results in:\n\nThe epistatic heritability (h2e) was set analogous to the additive heritability (h2), such that H2 = h2 + h2e. The additive × additive epistasis model was used, with only a single pairwise interaction. The epistatic effect βe was sampled from N(0, √h2e) and attributed to a single interacting pair of markers (e1, e2) such that βe = βe1 × βe2. We sampled this interacting pair from the set of additive QTNs; therefore, each interacting marker will always have some main effect. As for additive phenotypes, we also created nonlinear phenotypes with one large effect QTN. The total number of settings (scenarios considered in Table 1) for the simulated GP characteristics was 135 per phenotype class, i.e. linear and nonlinear. For each class, phenotypes were simulated with and without a large effect QTN. Thus, in total 810 (135 × 2 × 3) simulated phenotypic scenarios were generated, each having five independent phenotypic traits.22,23 These will be referred to as ‘simdata’ in the text.\n\n* Note: 1k=1000.\n\nReal datasets\n\nTo compare trends observed in simulations with outcomes obtained with real traits, publicly available wheat genotype and phenotype data were taken from Norman, Taylor.24 This includes 13 traits: biomass, glaucousness, grain protein, grain yield, greenness, growth habit, leaf loss, leaf width, Normalised Difference Vegetative Index (NDVI), physiological yellows, plant height, test weight (TW) and thousand kernel weight (TKW). This particular dataset was chosen as it contains a fairly large number of genotypes (n = 10,375) each genotyped for p = 17,181 SNPs. The impact of population structure, training set size, marker density and its interaction with population structure was assessed in a study by the same authors25 and GBLUP prediction accuracies were reported to saturate when training set size was greater than 8,000. We used the same settings, with five-fold cross-validation repeated for five times (training set size 8,300, validation set size 2,075).\n\nThe data was generated from a small-plot field experiment for pre-screening of germplasm containing some genotypes that are sown in multiple plots, thus containing spatial heterogeneity with correlation between closely located plots and imbalance in the number of phenotypes per genotype. Soil elevation and salinity, spatial coordinates and virtual blocks (made available on request by the authors) were taken as covariates:\n\nHere, X is the n × 4 design matrix for the fixed effects and overall mean, b is a 4 × 1 vector of fixed effects, i.e. soil salinity and elevation; Z is an n × 3 design matrix for non-genetic random effects u, i.e. range, row and block; Zg is the n × k design matrix for genotypes g for a maximum of k replicates, and ε is an n × 1 vector of residuals. The Best Linear Unbiased Estimates (BLUEs) of genotypes were used for GP; in this way, we take care of the experimental design factors. Note that equation (3) does not contain any SNP information, instead only genotype accessions are used to obtain their adjusted phenotypes.22,23\n\nTo analyse the influence of population structure on the performance of different GP methods, we used a population of the Arabidopsis thaliana RegMap panel26 with known structure, containing 1,307 accessions including regional samples (Extended Data, Figure S627). Linear phenotypes were simulated using narrow-sense heritabilities h2 = 0.1, 0.4 and 0.7, with equal effect QTNs. The genotypes, available from the Arabidopsis 250k SNP array, were further pruned for LD and minor allele frequency (MAF > 5%) using PLINK v1.9 (RRID:SCR_001757).28 LD pruning was carried out using a window size of 500 markers, stride of 50 and pairwise r2 threshold of 0.1, using the ‘--indep-pairwise’ command. This implies that a set of markers in the 500-marker window with squared pairwise correlation greater than 0.1 is greedily pruned from the window until no such pairs remain. This dataset will be referred to as ‘STRUCT-simdata’ in the text.22,23\n\nThe effect of population structure was also assessed on real data: a genotype dataset of 300 out of the 1,307 RegMap accessions, phenotyped for the sodium accumulation trait with a strongly associated gene.29 This should resemble one of our simulation scenarios, i.e. high heritability (e.g. h2 = 0.7) with few QTNs (e.g. 5) of large effect. This dataset will be referred to as ‘STRUCT-realdata’ in the text.22,23\n\nTo correct for population structure, we used principal components corresponding to the top ten highest eigenvalues as fixed effects in the models for GBLUP, RKHS regression, BayesA and BayesB.30 Principal component analysis (PCA) was performed on the allele dosage matrix using the prcomp() method in R, with centering and scaling. For random forest and XGBoost, we used these top principal components as additional features in the models.\n\nTo explore the impact of varying LD between SNP markers and actual QTNs on the performance of GP methods, we used two other datasets: one with real genotypes and simulated phenotypes, the other with real genotypes and real traits.\n\nFor the first dataset, we selected a natural population with minimal structure, balanced LD, genotyped at roughly equal genomic spacing and mostly inbred lines: the 360 accessions in the core set of the Arabidopsis thaliana HapMap population.29 Genotype data of 344 out of the 360 core accessions was obtained from Farooq, van Dijk,31 containing 207,981 SNPs. The phenotypes were simulated using one of the scenarios in the Section ‘Simulations’. The total number of SNPs was kept close to the number of samples and genetic complexity was kept low, to study the impact of SNP-QTN LD only. To this end, we simulated linear phenotypes with h2 = 0.7 and 5 QTNs with equal effects. Linkage disequilibrium between SNPs was calculated as squared pairwise Pearson correlation coefficient (r2) using PLINK v1.9 (RRID:SCR_001757).28 Input sets of 500 SNPs were selected randomly from pairs with either low LD (r2 ≤ 0.5) or high LD (r2 > 0.9); these two sets were used to train two prediction models using each GP method: one model was trained on the QTNs that were used to generate the phenotype, another on QTN-linked SNPs (closest on the genome) instead of the QTNs themselves, from the low or high LD SNPs pool. To avoid spurious correlations between SNPs in both models, non-QTN-linked SNPs were sampled from a different chromosome. We restricted the sampling of QTNs and the QTN-linked SNPs to chromosome 1, whereas the remaining non-QTN SNPs were sampled from chromosome 2. We refer to this dataset as ‘LD-simdata’ in the text.22,23\n\nFor the second dataset, we used three soybean traits (HT: height, YLD: yield and R8: time to R8 developmental stage) phenotyped for the SoyNam population.32 This dataset contains recombinant inbred lines (RILs) derived from 40 biparental populations and the set of markers have been extensively selected for the above traits. Moreover, high dimensionality is not an issue as the dataset contains 5,014 samples and 4,235 SNPs. We refer to this dataset as ‘LD-soy’ in the text. A complete list of datasets used in this study has been provided in Table 2 and achieved into public repositories.22,23\n\nA wide range of statistical models have been proposed for GP. Most widely applied are Linear Mixed Models (LMMs), which use whole-genome regression to tackle multicollinearity and high-dimensionality with shrinkage during parameter estimation, employing either a frequentist approach, e.g. restricted maximum likelihood (REML), or Bayesian theory.33 Below, we briefly describe the GP methods used in our experiments. For (semi) parametric methods, we used BGLR v1.1.0 (RRID:SCR_022522) with default settings of hyperparameters34; for Random Forests, the ranger R package v0.14.1 (RRID:SCR_022521)35; and for XGBoost, h2o4gpu v0.3.3 (RRID:SCR_022520).36\n\nParametric models\n\nGBLUP\n\nThe genomic best linear unbiased prediction (GBLUP) method uses a Gaussian prior with equal variance for all markers and a covariance matrix between individuals, called the genomic relationship matrix (GRM), calculated using identity by state (IBS) distances between markers for each pair of samples.37 SNP effects are modelled as random effects that follow a normal distribution with zero mean and common variance, and are estimated by solving the mixed model equation:\n\nHere, g is an n × 1 vector of the total genomic value of an individual, captured by all genomic markers; μ is the overall population mean; and ε is an n-vector of residuals. The genomic values g and residuals were assumed to be independent and normally distributed as g ~ N(0,Gσ2g), ε ~ N(0,Iσ2ε). Here G is the GRM, calculated using the rrBLUP v4.6.1 (RRID:SCR_022519) package38 in R, providing variance-covariance structure for genotypes and I is the identity matrix. Due to the small number of estimable parameters, GBLUP is computationally fast but the assumption of normality only holds when most effects are close to zero and only a few are larger. The limitation of this approach is that it captures only linear relationships between individuals and assumption of equal variance for all marker effects may not be truly valid for many traits.\n\nBayesian methods\n\nSeveral Bayesian methods with slight variations in their prior distributions have been proposed to model different genetic architectures39 e.g. BayesA, using a scaled t-distribution; Bayesian LASSO or BL,40 using a double-exponential; BayesCπ41 and BayesBπ,1 both utilising two-component mixture priors with point mass at zero and either a Gaussian or scaled t-distribution, respectively. To control the proportion of zero effect markers, the hyperparameter ‘π’ was set equal to 0.5, resulting in a weakly informative prior. For simplicity, we refer to BayesBπ as BayesB in the text. The model in equation (5) was solved for posterior means in both BayesA and BayesB with the only difference in priors of βj:\n\nHere, μ is the intercept, xj is an n-vector of allele dosages for each SNP and βj is the effect of SNP j out of a total of J SNPs.\n\nSemi-parametric models\n\nReproducing Kernel Hilbert Spaces (RKHS) regression is a general semiparametric method that models pairwise distances between samples by a Gaussian kernel and can therefore better capture nonlinear relationships than GBLUP. In fact, GBLUP is a special case of RKHS regression, with a linear kernel42,43 https://paperpile.com/c/ZyQHHy/1oKK+NO1v. We used RKHS regression as a representative semi-parametric model, because it not only employs prior assumptions for random components in LMM equation (6), but also learns hyperparameters from the data itself:\n\nIn contrast to the GBLUP model (4), the RKHS regression model has three random genetic components g=∑l=13gl, such that gl ~ N(0,Klσ2gl); where Kl is the kernel evaluated for the lth component. This kernel matrix K is used as genomic relationship matrix, where K = {k (xi, xj)} is an n × n matrix of Gaussian kernels applied to the average squared-Euclidean distance between genotypes:\n\nThe kernel k (xi, xj) is a covariance function that maps genetic distances between pairs of individuals xi and xj onto a positive real value. The hyperparameter b, called the bandwidth, controls the rate at which this covariance function drops with increasing distance between pairs of genotypes. Tuning this parameter for range of values between 0 and 1 could be computationally inefficient. So, instead of tuning b, we used a kernel averaging method,42 such that multiple kernels, corresponding to possible bandwidth values b = {0.2, 0.5, 0.8}, were averaged.\n\nEnsemble machine learning models\n\nRandom Forest\n\nThe Random Forest (RF) regressor uses an ensemble of decision trees (DTs) that are each grown using bootstrapping (random sampling with replacement of samples), and a random subset of SNPs. The test sample prediction is made by averaging all unpruned DTs as;\n\nHere x is the test sample genotype using an RF τ with D decision trees, for which ψk is the kth tree. An RF has a number of hyperparameters that need to be tuned, for which we used grid search using the caret v6.0.92 (RRID:SCR_021138) R package44 https://paperpile.com/c/ZyQHHy/GaA1. We used 500 trees in the forest for all analyses and tuned ‘mtry’ and ‘nodesize’ hyperparameters to control tree shapes. The total number of SNPs randomly selected at each tree node, i.e. mtry, was selected from {p/3, p/4, p/5, p/6} and the minimum size of terminal nodes below which no split can be tried, i.e. nodesize, was selected from {0.01, 0.05, 0.1, 0.2, 0.3} times the number of training samples in each cross-validation fold.\n\nExtreme Gradient Boosting (XGBoost)\n\nWe used XGBoost, a specific implementation of the Gradient Boosting (GB) method. Similar to the Random Forest, Gradient Boosting is an ensemble method, using weak learners such as DTs. The main difference is that an RF aggregates independent DTs trained on random subsets of data (bagging), whereas GB grows iteratively (boosting) by selecting samples in the subsequent DTs based on sample weights obtained in previous DTs, related to how well samples are predicted already by these previous DTs.\n\nHyperparameters were tuned using a grid search through five-fold cross-validation on each training data fold. We searched over max_depth = {2, 3, 4, 50, 100, 500}, colsample_bytree = {0.1, 0.2, 0.3, 0.5, 0.7, 0.9} and subsample = {0.7, 0.8, 0.9}.\n\nPerformance evaluation\n\nModel performance was evaluated based on prediction accuracy, which was measured as the Pearson correlation coefficient (r) between observed phenotypic values and predicted genomic values of the test population. For each model, five repeats of five-fold cross-validation were performed, so in total 25 values of r were used to compare performances. Statistical comparison between different models was performed by comparing prediction accuracies of each pair of models as a whole, i.e. on all values of p/n together using Wilcoxon rank-sum test.\n\nTo link GP performance in simulation scenarios with performance on real data, an assessment of the nature of real traits (i.e. linear or nonlinear) was used. To obtain a proxy for linearity of the trait, we assumed that if a trait has a higher proportion of additive variance compared to other traits, estimated with the same model, it will be more linear. To verify this on our simulated dataset scenarios (Table 1) for nonlinear phenotypes, we used the linear mixed model:\n\nHere ga defines a set of additive genotype effects such that ga ∼ N(0,σ2aG), where G is the genomic relationship matrix (GRM) calculated as described by VanRaden,37 gr ∼ N(0,σ2rI) is the residual genetic effect and ε is a vector of residuals. The ratio of additive genetic variance to the residual genetic variance (σ2a/σ2r) was calculated for both the simulated dataset and real wheat traits. We tested our assumption on simulated phenotypes (Extended Data, Figure S127), showing simulated amounts of non-additive heritability to indeed be negatively related to empirical additive heritability.\n\n\nResults\n\nPreviously, numerous GP methods were tested for different traits of varying genetic architectures using low or high density marker sets, but it is still unclear for which (class of) GP problems applying machine learning (ML) can be beneficial.9 To investigate the role of underlying characteristics (Figure 1), we generated an extensive set of simulated genotype-phenotype data (simdata: see Section ‘Simulations’). This data was analysed using the linear parametric methods GBLUP, BayesA and BayesB; the nonlinear semi-parametric regression method RKHS, using a Gaussian multi-kernel evaluated as average squared-Euclidean distance between genotypes42; and popular nonlinear ML methods, i.e. support vector regressor (SVR), random forest regressor (RF), extreme gradient boosting (XGBoost) regression trees and a fully-connected feed forward artificial neural network i.e. Multilayer Perceptron (MLP). The simulations covered a variety of trait scenarios (from simple to more complex), as shown in Table 1. Simple oligogenic traits correspond to simulation scenarios with larger heritabilities, additive allele effects (linear) and small numbers of QTNs; complex traits can have both additive and non-additive allele effects (nonlinear) with small heritabilities and large numbers of QTNs. For linear phenotypes, narrow-sense heritability was set equal to broad-sense heritability and for the nonlinear phenotypes, the sum of narrow-sense and epistatic heritability was set equal to the broad-sense heritability. The extent of phenotypic linearity in both simulations and real datasets was calculated using the ratio of additive genetic variance to the residual genetic variance (σ2a/σ2r) from equation (9). In the results presented below, SVR and MLP were excluded because their performances were significantly lower than the tree-based ensemble ML methods (i.e. RF and XGBoost) on a subset of our simulation scenarios (Extended Data, Appendix I45). Moreover, the applicability of neural networks/deep learning for GP in the feature space is still limited due to their high tendency toward overfitting under high-dimensionality until they are properly regularized or feature selection is employed.16,46,47\n\nML methods perform well for simple traits\n\nMany non-mendelian plant traits are fairly simple, where only one or a few QTLs explain a large proportion of phenotypic variance, called oligogenic traits. If these QTLs are identified by the GP model, prediction performance can be pretty high. In our simulations (Table 1), this scenario is investigated using additive phenotypes with narrow-sense heritability (h2) equal to 0.7 and a total number of QTNs equal to 5. We then alternatively attribute equal effects to all QTNs, assign a larger effect to the first QTN in equation (1) compared to other the QTNs, or sample the QTN effects from a Gaussian distribution (see Section ‘Simulations’).\n\nThe results in Figure 2A, Figure 2B and Figure 2C illustrate that the performance of Bayesian methods and ML was significantly better (p value < 0.01; Extended Data, Table S148) than that of genomic relationship-based methods (GBLUP, RKHS). The performance of ML methods was slightly poorer than that of Bayesian methods when all QTNs effects were equal (Figure 2A) or sampled from a Gaussian distribution (Figure 2C) but comparable when one of them had a larger effect size (Figure 2B). Therefore, although not outperforming the other methods, ensemble ML methods seem to be reasonable choices for simple traits.\n\nPerformance of parametric (GBLUP), semi-parametric regression (RKHS), parametric Bayesian (BayesA, BayesB) and nonparametric ML (RF and XGBoost) methods as average accuracy over 5-fold cross-validation of test data. Here accuracy is defined as Pearson correlation coefficient between true and predicted values. Each panel is a subset of the simulated scenarios in ‘simdata’ for a particular heritability and #QTNs. The ratio of the number of markers to the number of samples (c = p/n) increases from left to right in each subplot. A) Simple traits, simulated as linear polygenic phenotypes with only additive effects such that #QTNs is equal to 5 and h2 is 0.7, using equation (1), with all QTNs having equal effects. The largest standard error of mean for all values of c for each of the model was 0.023, 0.018, 0.007, 0.008, 0.018 and 0.009 for GBLUP, RKHS, BayesA, BayesB, RF and XGBoost respectively; B) similar to A, except one of the QTN had a large effect than others. The largest standard error of mean for all values of c for each of the model was 0.022, 0.022, 0.006, 0.007, 0.006 and 0.008 for GBLUP, RKHS, BayesA, BayesB, RF and XGBoost respectively; C) similar to A and B, except QTN effects were sampled from a Gaussian distribution; D) Complex traits, simulated as nonlinear polygenic phenotypes with both additive and epistatic effects such that #QTNs equal to p/2 and h2 is equal to 0.4, using equation (2), such that all QTNs had equal additive effects. Two of the QTNs were attributed to the epistatic effect such that Broad-sense heritability was set to 0.8 (H2 = h2 + h2e = 0.8). The largest standard error of mean for all values of c for each of the models was 0.03; E) similar to D, except one of the QTN had a large effect than others; F) similar to D and E, except QTN effects were sampled from a Gaussian distribution (see methods).\n\nML methods outperform parametric methods for complex traits\n\nComplex polygenic traits may contain a large effect QTL along with many small to medium effect QTLs.49 Despite assuming perfect LD between SNPs and their corresponding QTLs, their detection remains challenging through conventional univariate regression models that are followed by strict multiple testing corrections. Moreover, shrinkage of random effects towards zero in multivariate regression models restricts them from growing too large. Thus, many true small effects may be ignored in the analysis. SNPs may also have non-additive effects, which could cause a large amount of variance to remain unexplained and narrow-sense heritabilities to be low, when modelled by their linear action only.\n\nThis genetic complexity was simulated by increasing the number of QTNs, decreasing the narrow-sense heritability and keeping overall effect sizes equal, thereby letting the effect sizes per QTN become proportionally smaller. The QTNs were randomly chosen from the simulated SNPs pool by setting k equal to half of the total number of SNPs (p/2) in equation (2), keeping equal effect sizes for all QTNs and h2 equal to 0.4. Moreover, similar to simple traits, the other two scenarios, i.e. unequal effect sizes and normally distributed effect sizes, were also simulated. Two QTNs were randomly selected to have a fairly large pairwise interaction effect, corresponding to an epistatic heritability h2e equal to 0.4. The results in Figure 2D illustrate that ML methods significantly outperformed all methods for complex nonlinear phenotypes when all of the QTNs had equal effects (p-value < 0.01; Extended Data, Table S248). Interestingly, when one of the QTN had a larger effect size or was attributed with most of the variance, the Bayesian methods performed on par with ML (Figure 2E), but when the effect sizes followed a Gaussian distribution (Figure 2F), ML was outperformed by the other methods. This confirms that parametric methods work well if the effects distribution matches the statistical prior assumptions. In reality, genetic variance may not be attributed to a single Gaussian for other than infinitesimal model, instead it could be decomposed into multiple distributions enriched in multiple chromosomal localisations defined by heritability models.50 This phenotype complexity is usually unknown and difficult to accurately assess, which provides room for the ML methods.\n\nML methods are generally suitable for nonlinear phenotypes\n\nFor complex phenotypes, we observed that ML outperformed LMMs under highly polygenic phenotypes with epistatic effect and equivalent to Bayesian LMMs when at least one QTN had larger effect (Figure 2D and E). To explore further, we investigated a range of additive and non-additive fractions of heritabilities, with or without a large effect QTN and from Gaussian distribution defined in our simulation scenarios (Table 1).\n\nFor linear phenotypes with equal QTN effect sizes, performance of ML methods was poorer than that of Bayesian methods under all scenarios; with an increase in genetic complexity (lowering h2 and increasing the number of QTNs), performance dropped below that of GBLUP and RKHS as well (Extended Data, Figure S2A27). Therefore, ML methods are not beneficial for this setting. For nonlinear phenotypes however, ML outperformed all methods including the Bayesian methods for all scenarios (Extended Data, Figure S2B27), with random forests generalizing the best. ML methods are thus best suited for nonlinear traits and do not necessarily need large main effects to be present. Note that although RKHS regression has been reported to better capture epistatic relationships between markers,43 it did not perform well in our simulations; perhaps it needs more careful tuning of the bandwidth of the Gaussian distributions, rather than using multi-kernel averaging or require matching prior allele effects distributions (see Discussion, ‘Tree-based ensemble ML methods are a reasonable choice for GP’).\n\nFor the phenotypes explained by a large effect QTN and many small effect QTNs (Extended Data: Figures S3A and S3B27), Bayesian methods perform comparable to ML methods for both linear and nonlinear phenotypes under all simulation scenarios, although RF gave slightly better performance for nonlinear phenotypes with large epistatic heritability (for h2e = 0.7) and dimensionality (p/n > 2). This could be because the large effect QTN explains most of the additive variance and is easily picked by Bayes and ML methods, but RF has the added advantage of picking up the nonlinear signal, when main effects got smaller with the increase in number of QTNs. XGBoost gave relatively poor performance, especially at smaller heritabilities (0.1 and 0.4) and larger p/n ratios, while GBLUP and RKHS regression performance was consistently poor in all scenarios.\n\nFor both linear and nonlinear phenotypes (Extended Data: Figures S4a, and S4b27), the ensemble ML methods were still superior over BLUPs and comparable to Bayes when effect sizes were sampled from a Gaussian distribution for a small number of QTNs (e.g. q = 5, h2 = 0.7, h2e = 0.1), but the advantage diminishes when q increases and approaches the infinitesimal model i.e. q = p.\n\nIn conclusion, our simulation results indicate that ML works well when a fair proportion of broad-sense heritability is contributed by nonlinear allele effects or a few large effect QTNs.\n\nML performance is robust to high-dimensional GP\n\nGenomic prediction is usually employed on a genome-wide set of markers to yield total genomic value, but the training population size is limited, i.e. a high dimensional problem. This results into more statistical power to detect QTLs with many SNPs in LD but comes with obscured genetic variance when added together. Consequently, it leads to an overestimation of allelic variances or genomic relationships, overfitting on training samples and reduced performance on unseen data. To investigate the susceptibility of different GP methods for this issue, we analysed how prediction accuracy varied depending on the ratio of markers vs samples (c = p/n > 1).\n\nIn general, the results with different simulation settings of ‘simdata’ for linear phenotypes show that performance is negatively related to an increase in dimensionality when main effects got smaller due to decreasing heritability or increasing total number of QTNs (Extended Data: Figure S2A, Figure S3A and Figure S4A27). This implies that for simple traits having one or few large effect QTNs (Figure 2A to C), performance degradation is not a severe issue for Bayesian and ML methods but it can still be a potential problem for genetic distance-based methods i.e. GBLUP and RKHS., presumably because of increased uninformative markers in calculating the genetic kinships. For the nonlinear phenotypes, high dimensionality still doesn’t affect ML until we have sufficiently large main effects (Figures 2A and 2B; Extended Data: Figure S2B, Figure S3B and Figure S4B,27). Here, for the case when main effects were sampled from a Gaussian distribution, increasing polygenicity is analogous to having many small main effects; so, despite having epistatic effects, performance goes down for all methods. In the nutshell, this shows that the conclusions drawn in Section ‘ML methods perform well for simple traits’ and Section ‘ML methods outperform parametric methods for complex traits’ holds under high-dimensionality.\n\nCase study in wheat\n\nTo see whether our simulation results hold on real traits, we used a dataset of 13 wheat traits24 for a fairly large number of samples (10,375 lines) and 17,181 markers (c ≈ 1.6). These markers have been selected by strict screening criteria, therefore, many of them could be informative. Insights in the genetic complexity for some of these traits were previously reported in Norman, Taylor24 and Norman, Taylor.25 For example, glaucousness was reported to be a simple trait, but grain yield to be more complex.25 The results in Figure 3 clearly indicate that five-fold cross-validated prediction accuracies (r) were higher for both ML methods when the fraction of additive variance was small (i.e. traits were fairly complex) and slightly lower or comparable to both Bayesian and GBLUP/RKHS regression methods otherwise. This is in line to what we observed in our simulations: for simple traits (Figure 2A and B) ML performance was either comparable to Bayesian or slightly poorer, but for complex traits it was consistently better (Figure 2C). For example, leaf width, glaucousness, growth habit, leaf loss, plant height, test weight and thousand kernel weight traits had greater than 80% of their genetic variance explained only by additive variance components and performance of ML relative to Bayesian methods and GBLUP/RKHS regression was either at par or lower than that. On the other hand, biomass, grain protein, grain yield, yellowness and in particular NDVI had smaller fractions of additive variance and, relative to the other methods, ML performed better. Hence, results on this experimental dataset match with the findings in our simulations that ML is best suited for the prediction of more complex traits and a potential candidate for simple traits as well.\n\nTop: prediction accuracies for GP models on wheat traits, reported as the mean Pearson correlation coefficient (r) of 5-fold cross-validation. Trait abbreviations are given in Table 2. Bottom: fraction of additive to residual genetic variance calculated using equation (9) for each trait. Traits were sorted in ascending order of additive variance fraction (left to right); therefore, the leftmost trait (NDVI) can be considered more complex than those to the right.\n\nPopulation structure (PS) is a well-known confounding factor that results in decreased diversity in training populations25 and unrealistic inflated parameter estimates, e.g. for (co) variances of random effects in LMMs51https://paperpile.com/c/ZyQHHy/ByqH. Parametric and nonparametric ML methods, based on their modelling assumptions and approaches, may be differently sensitive to PS. To assess the impact of population structure on ML methods, we used real genotype data with a known population structure and combined it with both simulated (STRUCT-simdata) and real phenotypes (STRUCT-realdata). Only linear phenotypes were simulated, with varying complexity and dimensionality scenarios, as described earlier in Section ‘Simulations’. The STRUCT-simdata contains all 1,307 Arabidopsis RegMap accessions.26 To exclude the impact of multicollinearity among SNPs, only uncorrelated markers were retained after pruning with pairwise squared correlation coefficient (r2 < 0.1, see Section ‘Population structure analysis’), leaving 15,662 SNPs, but keeping the population structure intact (Extended Data, Figure S727). This results in a ratio c = p/n of approximately 12 (15,662/1,307), a setting comparable to the simulation results presented in Figure 2A.\n\nCorrection for PS was carried out by including the top ten principal components corresponding to the largest eigenvalues as fixed effects into the mixed model equations or as additional features for ML methods. For the simulated phenotypes (Extended Data, Figure S627), average pairwise difference of test accuracies before and after correcting for PS was slightly higher for ML methods (RF: 0.03 and XGBoost: 0.04) than for LMMs (GBLUP: 0.01, RKHS: 0.01, BayesA: 0.01 and BayesB: 0.00). Moreover, the correction resulted into relatively elevated accuracies for the scenarios with larger number of QTNs or low heritabilities. This illustrates that with smaller #QTNs and larger heritabilities (h2 = 0.7, #QTNs = 5), effect sizes per QTN were larger; therefore, confounding due to PS was less of a concern. With the decrease in effect sizes per QTN (increase in #QTNs and decrease in h2), correction became more important for reliable predictions. From this, we can argue that confounding due to PS should be generally corrected for, but particularly for complex phenotypes having low heritability and large numbers of QTNs with small-medium effect sizes.\n\nTo further explore this behaviour, we used real phenotypes of the sodium accumulation trait in Arabidopsis thaliana (STRUCT-realdata) using a subset of the same genotypes dataset. Here, we expected to have at least one large effect QTN for this trait, because AtHKT1;1 locus, encoding a known sodium (Na+) transporter, has been reported to be a major factor controlling natural variation in leaf Na+ accumulation capacity.29 Similar to the outcomes on ‘STRUCT-simdata’, correction for PS increased prediction accuracies of all methods on test data; whereas, GBLUP showed the lowest average difference (∆μ = 0.03) in performance before and after correction (Figure 4). In contrast to ‘STRUCT-simdata’, XGBoost had the largest average difference (∆μ = 0.1) but for RF the difference was comparable to LMMs (∆μ = 0.05). From the above outcomes, we conclude that ML methods, like other GP methods, are sensitive to confounding due to PS and correcting for this can further improve performance for complex phenotypes. However, it is still unclear to which extent or for which GP problem characteristics different methods are more advantageous or more sensitive to PS.\n\nBoxplots present Pearson correlation coefficients (r) found in 5-fold cross-validation, on test data from ‘STRUCT-realdata’. Here ∆μ is the average difference between pairwise predictions before and after correction and for each model, the nonparametric Wilcoxon rank sum test was used to assess statistical significance.\n\nThe utility of GP in genomic selection is based on the assumption that there are ample markers within a densely genotyped set of markers which are in LD with the QTLs.1 The actual QTNs are generally unknown, but SNPs in LD can be used to (partially) capture their effect, depending on the actual correlation and allele frequencies. Therefore, it is worthwhile to investigate the impact of SNP-QTN correlation levels on GP performance52https://paperpile.com/c/ZyQHHy/Q1iWE. We used two settings, one with real genotypes and simulated phenotypes (LD-simdata), a second with real genotypes and real traits (LD-soy).\n\nIn simulations, GP model performance is evaluated based on the difference in prediction accuracies between a model trained on the actual QTNs and a model trained on SNPs in LD (QTN-linked SNPs). Our results show that when SNPs are highly correlated to QTNs (which is likely the case for densely genotyped markers set and r2 > 0.9), all methods perform equally well and the SNP-based model predictions are very close to those of the actual QTN based models (Extended Data, Figure S827). On the other hand, for low LD between SNPs and QTNs, there was in general a difference between median prediction accuracies (Δr) of the QTN and SNP-based models (Figure 5A). This difference varied between methods, from 0.18 for RKHS regression to 0.43–0.46 for the Bayesian methods, with GBLUP and ML methods between these (0.32–0.37). The relative robustness of particularly the Random Forest model in these circumstances compared to the Bayesian methods, in combination with its good performance in many simulations, supports its usefulness for GP.\n\nPrediction accuracy of different GP methods on simulated (A) and real soybean (B) datasets for high and low LD between SNPs and actual QTNs. The difference in median accuracies between these scenarios is indicated as Δr. A) LD-sim data, low SNP-QTN LD (r2 ≤ 0.5). B) LD-soy data, low (r2 ≤ 0.5) SNP-QTN LD vs. all SNPs (high LD).\n\nAs a real genotype and phenotype dataset, we used three Soybean traits, i.e. height, time to R8 developmental stage and yield (LD-soy). The complete set of markers (4,235 SNPs) had many correlated SNPs, such that only 261 were left with low LD (r2 ≤ 0.5). Here, in contrast to LD-simdata where we knew the QTNs in advance, we assumed that many SNPs could be linked to QTNs, because ~94% of all markers had r2 > 0.5. So, we compared two models: one with all markers (the benchmark model), and one with low LD (r2 ≤ 0.5). A similar pattern was observed, as shown in Figure 5B, i.e. RKHS regression, RF and XGBoost were most robust against low SNP-QTN LD, with negligible differences between median accuracies, where GBLUP and the Bayes methods had higher differences.\n\nIn conclusion, GP methods that model SNP-QTN or SNP-SNP relation as a nonlinear function (RKHS, RF, XGBoost) were more stable under low SNP-QTN LD compared to other methods (GBLUP, BayesA, BayesB). Moreover, RF seems to couple good prediction performance with reliability under low SNP-QTN LD.\n\n\nDiscussion\n\nGenomic prediction has long been the realm of parametric methods, but recently nonlinear supervised ML methods have become increasingly popular. Yet literature is unclear on the characteristics of GP problems that warrant application of ML methods. This study fills this gap and concludes that nonlinear tree-based ensemble ML methods, especially Random Forests, can outperform traditional methods for simple as well as complex polygenic traits where nonlinear allele effects are present. Moreover, ML methods are robust to high dimensionality, although further improvements, e.g. statistical or prior knowledge driven regularization, may improve performance. ML methods are particularly useful compared to the frequently used GBLUP and RKHS regression given their higher performance. While Bayesian methods often perform on par with ML models, this is mainly when there are large effect QTNs and/or linear phenotypes. Moreover, Bayesian methods are prone to overfitting in case of small sample sizes (p/n > 1), which is less of an issue with ML, especially with RF (Extended Data: Figures S9A and S9B27).\n\nA wide range of parametric, semi-parametric and nonparametric methods can be used for GP, but it is impractical to test all for a particular application. The choice for a suitable method strongly depends on the GP problem characteristics, described in Figure 1. While GP methodology can be compared using various model evaluation metrics (BIC, AIC, log likelihoods), we focused on their utility from a breeder’s perspective, so we compared only their prediction accuracies. We found that GP methods based on modelling the distance between genotypes using covariance structure(s), inferred from genomic markers (GBLUP and RKHS), were generally inferior to Bayesian and ML methods and less robust to high-dimensional problems likely because all of the p SNPs were used always to calculate the kinship matrices, whereas, either 5, 50, 100, p/2 or exactly p SNPs were chosen as QTNs. When q is fairly less than p, makes the kinship matrix too noisy due to the large number of markers that are unrelated to the phenotype but are used in the calculation of the GRM. Hence, we expect equal accuracies for increasing number of QTNs (q), keeping the other factors (p, n and h2) fixed. Figure S5 (Extended Data27) clearly illustrates that these methods indeed have constant prediction accuracies with increasing q values, while the accuracies of the other methods drop due to decreasing effect sizes. This further explains that their performance can be improved by removing unrelated markers from the GRM, for instance using biological knowledge about markers.31,53\n\nThe parametric LMM equations can be solved using a Bayesian framework. Bayesian methods define prior SNP effects distributions to model different genetic architectures. Instead of a single distribution for all marker effects (e.g. BRR), it could be defined for each individual marker (e.g. BayesA). Mixture distributions have also been proposed (e.g. BayesC, BayesB). From the Bayesian alphabet, we used BayesA and BayesB as representatives because the first scenario, i.e. a single distribution for all markers, has been covered by GBLUP. Our results illustrate that these methods outperform GBLUP and RKHS regression when large effect QTNs are present, for both linear and nonlinear phenotypes. On the other hand, tree-based ensemble ML methods had either comparable performance to Bayesian methods (for simple traits) or superior performance (for complex traits). Capitalising on the results from Appendix-I (Extended data27) that these ML methods had better performances than other ML methods (SVR and MLP), we can argue that these tree-based ML methods are a reasonable choice to conduct GP.\n\nPopulation structure can affect GP performance. Our results show that without correcting for population structure, test accuracies were lower than after correction for all methods. However, ML seems to be slightly more sensitive because the average difference between each pairwise test data accuracies was higher than other methods in the simulated data.\n\nConfounding due to population structure can also be due to the frequently employed random cross-validation strategy for predictive modelling.25 In random cross-validation, the reference population is randomly divided into subsets, one of which is iteratively selected for testing while the remaining subsets are used to train the model. While samples are all part of a test set once, under population structure some subpopulations may be over or under-represented in the training set. As a result, the model may get overfitted. A solution could be to use stratified sampling instead. On the other hand, parameter estimation may get misguided by within subgroup allele frequency differences rather than the overall true phenotype associated variance.\n\nThe impact of population structure can be dealt with in many ways. Conventionally, principal components of the SNP dosages or genomic relationship matrix are introduced as fixed effects in the mixed model equations.54–56 Alternatively, phenotypes and genotypes can be adjusted by the axis of variations before predictive modelling.5 Nevertheless, some residual structure often remains in the datasets, so it is important to check sensitivity of GP models to this confounding factor. Since ML methods (RF and XGBoost) do not employ any statistical prior and learn the association patterns from the data itself, they may be more sensitive to structure, as we found in our simulation results. But this is not clearly evident from the real phenotypes, so we cannot generalize this conclusion from our simulations.\n\nDespite technological improvements, low density SNP panels are usually cost-effective for routine genomic selection. Increasing marker density does not necessarily increase prediction accuracy, since accuracy is not a linear function of SNP density only.57–59 Instead, many GP problem characteristics (Figure 1) jointly affect performance. However, using low density SNP panels can negatively affect prediction performance, since relevant SNPs in LD with the QTLs can either be completely missing or SNPs only in low LD may be present. As a result, allele frequencies between SNPs and QTNs can be quite different, resulting in incorrect predictions.52 Despite this, low SNP density can still be sufficient for populations with larger LD blocks, e.g. F2 populations, where QTL detection power is highest and in this case, we shouldn’t expect much improvement by increasing marker density. But it becomes an important consideration when LD starts to decay and population relatedness decreases in the subsequent crosses of the breeding cycle. In this context, our study addresses the question of whether certain GP methods, especially ML, are more sensitive to low SNP-QTL LD. The results using both simulated and real traits indicate that SNP-QTL LD could also be an important determinant of suitable GP methodological choice and that ML is robust against low LD.\n\nA weak SNP-QTL correlation implies that the SNP is a weak predictor of phenotype and there is an imperfect match between the genotypic distribution and the actual underlying genetic distribution of the phenotype. When using penalized regressions, this can result in different shrinkage for the SNP than that required by the actual QTN, thereby leading to a low genetic variance attribution to that SNP. Therefore, we may expect better prediction by nonparametric ML methods, as they may better learn weak genetic signals and are more robust to low SNP-QTL LD problems. On the other hand, the semiparametric RKHS regression method, which measures genetic similarity between individuals by a nonlinear Gaussian kernel of SNP markers, also performed better than GBLUP and Bayesian methods under low SNP-QTN LD. The reason could be that under low SNP-QTN LD, true pair-wise genetic covariance estimation would be less accurate due to losing many important markers and considering all of them equal contributors towards total genetic covariance. In case of RKHS regression, a Gaussian distribution defines a SNP’s probable contribution towards total genetic covariance, which becomes more realistic in this scenario because fewer important SNPs are left than in the high SNP-QTN LD case. The Bayesian methods (BayesA and BayesB) had the largest decrease in test performance under low SNP-QTN LD compared to high SNP-QTN LD. This could be due to the application of penalties on individual marker effects, which shrinks the weak SNP-QTN associations towards zero for each SNP.\n\nBread wheat breeding has huge impact on worldwide food security and socio-economic development.60 Therefore, minor improvements in GP methodology leading to overall genetic gain can have high impact. In this study, we used a large (10,375 lines) Australian germplasm panel, genotyped with a high quality custom AxiomTM Affymetrix SNP array and phenotyped for multiple traits with varying complexity levels.24 The authors showed that genomic selection was superior to marker-assisted selection (MAS) by employing GBLUP with two random genetic components (referred to as full-model in their text). Our results clearly indicate that ML can perform well for complex bread wheat traits, e.g. grain yield, yellows, greenness, biomass and NDVI. All of these traits except grain yield can be measured using high-throughput automated phenotyping.61 This is an interesting finding since, with the rapid advances in low cost high-throughput phenotyping systems, attention is shifting towards measuring component traits, e.g. vegetative indices, rather than final yields. ML methods can predict these traits more accurately, as evident from our analysis.\n\n\nConclusions and outlook\n\nBased on simulated and real data, we conclude that tree-based ensemble ML methods can be useful for GP for both simple and complex traits. Moreover, these methods can work for both low- and high-density genotyped populations and can be a first choice for practical plant breeding. However, proper correction for population structure should be applied to obtain stable accuracies. Between bagged (Random Forests) or boosted (XGBoost) decision tree ensemble methods, Random Forests seem to be a good first choice for GP given their generalization performance and ability to work with high dimensional genotype data. It would be interesting to investigate to what extent these ML methods can benefit from statistical or prior knowledge-based regularization techniques.\n\n\nData availability\n\nAll datasets analysed during the current study are already published and publicly available22,23 and references to their authors or repositories have been mentioned in the text.\n\nFigshare: Extended data for ‘Genomic prediction in plants: opportunities for ensemble machine learning based approaches’.\n\nThis project contains the following extended data:\n\n‐ Supplementary Figures: http://www.doi.org/10.6084/m9.figshare.1991900225\n\n• Figure S1. Assessment of phenotypic class (linear or nonlinear).\n\n• Figure S2. Comparison of test data prediction performance using simulated phenotypes with equal effects QTNs.\n\n• Figure S3. Comparison of test data prediction performance using simulated phenotypes with unequal effects QTNs.\n\n• Figure S4. Comparison of test data prediction performance using simulated phenotypes with QTN effects sampled from Gaussian distribution.\n\n• Figure S5. Effect of increasing number of QTNs to the total number of SNPs ratio on prediction performances using simulated phenotypes linear phenotypes.\n\n• Figure S6. Effect of population structure correction on GP model accuracies.\n\n• Figure S7. Principal Component Analysis (PCA) of Arabidopsis thaliana RegMap 1,307 accessions using uncorrelated set of markers.\n\n• Figure S8. Effect of high SNP-QTN LD (r2>0.9) on prediction accuracy.\n\n• Figure S9. Comparison of training data prediction performances using simulated phenotypes with one large effect QTN.\n\n• Figure S10. Comparison of prediction performances of parametric, semi-parametric and ML methods using simulated phenotypes without a large effect QTN for nonlinear phenotypes.\n\n‐ Supplementary Tables: http://www.doi.org/10.6084/m9.figshare.1991872946\n\n• Table S1. Simple Traits\n\n• Table S2. Complex Traits\n\n• Appendix 1: Selection of machine learning (ML) candidates for genomic prediction.\n\nhttp://www.doi.org/10.6084/m9.figshare.1991902343\n\n\nSoftware availability\n\nSource code available from: https://git.wur.nl/faroo002/pub2\n\nArchived at the time of publication: https://doi.org/10.5281/zenodo.6734259.23\n\nLicense: GPL version 3",
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PubMed Abstract | Publisher Full Text\n\nBermingham ML, Pong-Wong R, Spiliopoulou A, et al.: Application of high-dimensional feature selection: evaluation for genomic prediction in man. Sci. Rep. 2015 May 19; 5: 10312. PubMed Abstract | Publisher Full Text | Free Full Text\n\nZhang A, Wang H, Beyene Y, et al.: Effect of Trait Heritability, Training Population Size and Marker Density on Genomic Prediction Accuracy Estimation in 22 bi-parental Tropical Maize Populations. Front. Plant Sci. 2017; 8: 1916. PubMed Abstract | Publisher Full Text | Free Full Text\n\nWang Q, Yu Y, Yuan J, et al.: Effects of marker density and population structure on the genomic prediction accuracy for growth trait in Pacific white shrimp Litopenaeus vannamei. BMC Genet. 2017 May 17; 18(1): 45. PubMed Abstract | Publisher Full Text | Free Full Text\n\nTechnow F, Riedelsheimer C, Schrag TA, et al.: Genomic prediction of hybrid performance in maize with models incorporating dominance and population specific marker effects. TAG Theoretical and Applied Genetics Theoretische Und Angewandte Genetik. 2012 Oct; 125(6): 1181–1194. PubMed Abstract | Publisher Full Text\n\nTessema BB, Liu H, Sørensen AC, et al.: Strategies Using Genomic Selection to Increase Genetic Gain in Breeding Programs for Wheat. Front. Genet. 2020 2020-December-04; 11(1538). English. PubMed Abstract | Publisher Full Text\n\nRabab S, Breen E, Gebremedhin A, et al.: A New Method for Extracting Individual Plant Bio-Characteristics from High-Resolution Digital Images. Remote Sens. 2021; 13(6): 1212. Publisher Full Text"
}
|
[
{
"id": "145166",
"date": "28 Sep 2022",
"name": "Muhammad Tehseen Azhar",
"expertise": [
"Reviewer Expertise Plant Breeding and Biotechnology",
"Genetics"
],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nAll the components are written according to international standards and latest information is cited. \nThe statistical designs are used according to the demand of hypothesis and nature of data for the interpretation of the results. The protocols are elaborated in this manuscript so that one can repeat these experiments for more interpretations. The needed statistical analyses are conducted for the interpretation of the data.\nMethods-Data-Simulations: Explain the reason why you selected 500 for the sample size. What was the rationale behind choosing fixed MAF=0.4 and not other than that?\nIn Figure 5B, why does yield have more accuracies than height and R8; whereas, yield is usually considered as a relatively more complex trait than others.\nThe data is submitted in the relevant repository and authors would be available to answer any query from the researchers.\nThe conclusion is to the point based on results and will be guideline for later studies. The Ref#55. Patterson N, Price AL, Reich D: Population Structure and Eigen analysis. PLoS Genet. 2006; 2(12): e190. should be removed because Ref#54 is its follow up study.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Partly\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nPartly\n\nAre all the source data underlying the results available to ensure full reproducibility? Partly\n\nAre the conclusions drawn adequately supported by the results? Yes",
"responses": [
{
"c_id": "9167",
"date": "10 Jan 2023",
"name": "MUHAMMAD FAROOQ",
"role": "Author Response",
"response": "We thank all reviewer for providing a valuable input to the article that helped us to improve it further. We have tried to address their comments one by one and made some changes to the text accordingly. Methods-Data-Simulations: Explain the reason why you selected 500 for the sample size. What was the rationale behind choosing fixed MAF=0.4 and no other than that? We chose 500 samples since a reference population of this size is common for many genomic selection applications in plant breeding, as mentioned on lines 112-113. For example, Nonoy et al., 2022 used 457 soybean breeding lines, Sandhu et al., 2021 used 650 spring wheat lines etc. We fixed MAF=0.4 for all SNPs, and decided not to incorporate the impact of allele frequencies because the MAF of QTLs can impact SNP heritability estimation and ultimately prediction accuracies (Yoshinobu Uemoto et al., 2015). With this setting, we obtained enough statistical power for each SNP during allele effects estimation (line 105-109). In Figure 5B, why does yield have more accuracies than height and R8; whereas, yield is usually considered as a relatively more complex trait than others. We used the ~4.2k SNPs extracted by Azodi et al., 2019 from the complete SoyNAM genotype dataset after rigorous feature selection. The motivation was to choose a real, low-dimensional dataset with highly correlated SNPs to understand the impact of SNP-QTL LD only, while benchmark accuracies were also available in that study. Note that in general the accuracy of a genomic prediction model depends on whether and how many of the SNPs causal for a trait are included in the model. It might be that the feature selection performed by Azodi et al. had a stronger impact on height and R8 compared to yield. In any case, our results do match with those of Azodi et al., 2019 (Figure 5A), who found lower accuracies for predicting height and R8 than for yield, given this selected subset. We thank the reviewer for pointing this out, and now explicitly mention this in the text on lines 636-638 and 645-646. The Ref#55. Patterson N, Price AL, Reich D: Population Structure and Eigen analysis. PLoS Genet. 2006; 2(12): e190. should be removed because Ref#54 is its follow up study. Thank you for pointing it out. This reference has now been removed."
}
]
},
{
"id": "153250",
"date": "20 Oct 2022",
"name": "Miguel Pérez-Enciso",
"expertise": [
"Reviewer Expertise Quantitative Genetics",
"Machine Learning"
],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nAuthors present an ample study on ML and linear methods for GP. Their conclusion is very clear (RF and XGBoost end to outperform linear methods); However, they overinterpret, - RF and related methods are better when a few variables are of large influence, as multiple studies have shown. It is true that ensemble methods tend to be a safe choice (see eg Azodi et al., 20191). As many studies have shown, the best method is trait and scenario dependent. Therefore, I suggest to include a small dose of caution in the conclusions. The paper is perhaps overlong.\nI am not sure a clear rationale on choosing between ML and LMM is lacking, but you do not solve it.\nThe term 'non linear phenotypes' is misleading, if it refers to the degree of non additivity, I am not sure we can tell so easily between linear and non linear phenotypes.\nA clear link between simulated and real phenotypes is often missing?\nThe simulation of allele frequencies is completely unrealistic; not detailed on how effects are sampled.\nTable 1: you use up to 60k QTLs?\nTable 2, provide also n and p.\nGBLUP can also include dominance and epistasis (work by Vitezica, Varona, et al., 20182).\nWhat are the 'Ith' components in equation 6?\nEquation 9, not clear how genetic residual is calculated nor its meaning? The estimated non additive variance?\nA negative relation by the way of additive and non additive part cannot be generalized.\nOut of all scenarios simulated, the most realistic is panel F (Fig 2), which shows similar behavior across methods, as usually observed.\nFig 3: weird some very big differences (e.g., NDVI phenotype).\n\nSome review refs\nAzodi et al. (2019)1 Reinoso-Peláez et al. (2022)3\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? Partly",
"responses": [
{
"c_id": "9168",
"date": "10 Jan 2023",
"name": "MUHAMMAD FAROOQ",
"role": "Author Response",
"response": "We thank the reviewer for constructive criticism and providing a valuable input to the article that helped us to improve it further. We have tried to address the comments one by one and made some changes to the text accordingly. Reviewer) I suggest to include a small dose of caution in the conclusions. I am not sure a clear rationale on choosing between ML and LMM is lacking, but you do not solve it. Authors) We agree that the choice of a particular method is scenario dependent. We also concur that despite simulating many scenarios, we do not find a definitive rationale for choosing between ML and LMM. Instead, our study could provide some general guidelines that can be helpful when considering this issue. We have revised the conclusion section to clarify this and modified the abstract to avoid over-interpretation. Reviewer) The term 'non-linear phenotypes' is misleading, if it refers to the degree of non-additivity, I am not sure we can tell so easily between linear and non-linear phenotypes. Authors) Thank you for pointing this out. Although we did define non-linear phenotypes as those exhibiting epistatic interactions, we agree the use of this term can be misleading. We have therefore replaced all uses of linear and non-linear phenotypes by additive and epistatic, respectively. Reviewer) The simulation of allele frequencies is completely unrealistic; not detailed on how effects are sampled. Authors) We fixed MAF=0.4 and decided not to incorporate the impact of allele frequencies in our analysis because MAF of QTLs can impact heritability estimation and ultimately prediction accuracies (Yoshinobu Uemoto et al., 2015). This allowed us to observe equal and reasonably enough statistical power for each SNP during allele effects estimation. To clarify for the readers, we have also mentioned this in the text now (lines 105-109). The effect sizes were sampled from a Gaussian distribution ~N(0, √ h 2), to simulate three different cases. For the first case, one effect β was sampled and then all q QTNs were allocated βi = β / q, i.e. all QTNs had equal effects. This allowed us to evaluate a trait complexity scenario where effect sizes could decrease with increasing numbers of QTNs. In the second case, we sampled two effect sizes (a large effect for a single QTN, a smaller effect for all other QTNs) from the effect size distribution. For the third case, all effects were randomly sampled from the Gaussian distribution. The Methods section now contains a more elaborate description of our approach on lines 130-141. Reviewer) Table 1: you use up to 60k QTLs? Authors) Yes; for the case in which we had 60k SNPs, one possible scenario was to use 60k QTNs. This was simulated to illustrate the infinitesimal modelling scenario. Reviewer) Table 2, provide also n and p. Authors) Thank you for the suggestion. Table 2 has been updated. Reviewer) GBLUP can also include dominance and epistasis (work by Vitezica, Varona, et al., 2018). Authors) Indeed it can, and there are many other variations. We believed a full comparison of these methods to be out of the scope of this manuscript. However we did use RKHS as a general class of BLUPs with a three-kernel averaging scheme (De los Campos et al., 2010) using bandwidth values b={0.2,0.5,0.8}. Reviewer) What are the 'Ith' components in equation 6? Authors) The RKHS method contains three random genetic effects. The ith component is the ith random genetic effect, with a genomic relationship matrix determined by Gaussian kernels corresponding to i = first, second or third value of bandwidth from {0.2,0.5,0.8}. We clarified this on line 294. Reviewer) A clear link between simulated and real phenotypes is often missing? Authors) We agree that a direct link between simulated and real traits is difficult to establish because simulations simplify things to a large extent. A better link could perhaps be established if the same population was utilised for simulated phenotypes. However, the purpose of our simulations was to give a general idea of where RF/XGB could be a potential choice, not for a specific population. We observed these methods work well for the case with a few QTNs with large effect or when the phenotype contains interaction effects. When the trait is highly polygenic and additive, such that the effect sizes distribution resembles a Gaussian (Figure 2), similar performance can be expected for RF/XGB and LMMs. Reviewer) Equation 9, not clear how genetic residual is calculated nor its meaning? The estimated non additive variance? A negative relation by the way of additive and non-additive part cannot be generalized. Authors) We agree that this was not clear and have changed equation (9) to accommodate the additive and epistatic random genetic components explicitly. Thus, the additive component is governed by the additive covariance matrix, whereas the epistatic component has a covariance matrix accommodating a fraction of the SNP interactions, as proposed by the E-GBLUP methodology (Yong Jiang and Jochen C Reif 2015). With both of these random components, we were able to extract the proportion of additive (σ 2a) and epistatic (σ2e) variances for a trait and therefore, the σ2a /σ2e , should in principle, be higher when epistatic variance is low and vice versa. We have mentioned this in the text for clarification on lines 349-355. This negative relation was also found in our simulated data (Figure S1). Accordingly, for the real wheat traits, we observed a negative relation between σ2a /σ2e and the maximum difference between LMMs and RF/XGB accuracies. There was a clear separation between the traits, as plotted in Figure R1, with a lower additive:epistatic variance ratio (e.g. GY, GP, NDVI, BM etc) and those with a higher ratio (e.g. TW, TKW, GL, PH, LW). We agree that this negative relation cannot be generalized, but given our results testing the modified equation (9) on multiple phenotypes of the same population, we believe it to be an extrapolation for the same population. In the above Figure R1, we can see that for traits that are predominantly additive and polygenic, LMMs and RF/XGB perform quite similar (as for the cases in Figure 2-panel F); but for traits with a predominant epistatic component, for example, grain yield and biomass, RF/XGB outperform LMM. Reviewer) Out of all scenarios simulated, the most realistic is panel F (Fig 2), which shows similar behavior across methods, as usually observed. Authors) We agree to some extent that F is more realistic for many complex traits, but the scenarios in panels B and C can also be observed, e.g. for oligogenic traits. An example is the sodium accumulation trait (Figure 4), where ML performed well due to the large effect QTN. The panels with equal effects were meant (as a theoretical exercise) to illustrate the impact of many small effect QTNs and small narrow-sense heritabilities. We have clarified this point further in the text on lines 658-666. Reviewer) Fig 3: weird some very big differences (e.g., NDVI phenotype). Authors) The large difference in prediction accuracy for grain yield and biomass could be attributed to their larger epistatic variance compared to the other traits. For the NDVI trait, phenotypic variance and narrow-sense heritability were low in this dataset, so this should be specific to this dataset only. We have explicitly mentioned this issue on lines 773-775 in the text."
}
]
}
] | 1
|
https://f1000research.com/articles/11-802
|
https://f1000research.com/articles/11-956/v1
|
18 Aug 22
|
{
"type": "Research Article",
"title": "The influence of strategic innovation management on firm innovation performance in the Indonesian mid-size telecommunication industry",
"authors": [
"Risris Rismayani",
"Bram Manuel",
"Umi Latifah",
"Bram Manuel",
"Umi Latifah"
],
"abstract": "Background: The telecommunication industry was one of the Indonesian government's priorities in the national development plan 2015-2035. “Primary Industry” was the term for the priority industries with the central role as the prime mover in the future national economy. Various natural, human, technological, innovative, and creative resources were imperative in supporting the underlining national industry development plan. Strategic innovation management refers to the entire sequence of innovation practices, including competition mechanism analysis, such as creating an innovative vision, business strategy alignment, disseminating strategy at an entire organizational level, market tendency, technology, and competitor’s action. Firm innovation performance refers to the measurement of innovation efficiency (the number of new products, product novelty, new product development speed, and new product success rate) and innovation profitability (new product revenue proportion, quality enhancement, cost reduction, and value improvement) conducted by the firm. This study investigates the effect of Innovation Strategy, Organizational Structure, Innovation Culture, Technological Capability, and Customer-Supplier Relationship (these were the practice of Strategic Innovation Management mentioned in various literature) on Firm Innovation Performance. Methods: A quantitative method, from a practical perspective, was employed to investigate the causal relationship between strategic innovation management and firm innovation performance. Data was gathered through a validated and reliable questionnaire disseminated to 90 respondents. It included a representative from the four sub-sectors of the telecommunication industry, namely fixed networks, wireless networks, telecommunication services, and special telecommunication. Results: The survey found that firms within the telecommunication industry already employed Strategic Innovation Management practices. Moreover, this study also found that Innovation Culture, Technological Capability, and Customer-Suppler Relationship significantly influence Firm Innovation Performance. Conclusion:\n\nThe implementation of Strategic Innovation Management in the mid-size companies within Indonesia's telecommunication industry appears to be relatively high. It indicates that firms within the industry were able to strategically compete by implementing Strategic Innovation Management.",
"keywords": [
"Firm Innovation Performance",
"Innovation Culture",
"Technological Capability",
"Customer and Supplier Relationship",
"Innovation Strategy",
"Organizational Capability"
],
"content": "Introduction\n\nThe telecommunication industry is one of Indonesia's most competitive and developing industries. It was claimed to be a prominent driver of the national economy.1 Therefore, it ultimately contributes to the efficiency and effectiveness of the National Budget, local industry, employment, national governance, and social development in science and telematics.\n\nIndonesia Ministry of Communication and Informatics stated in 2019 that the number of significant telecommunication firms in Indonesia was 216 entities.2 Their service entails wired telecommunication service, the Public Switched Telephone Network (PSTN), and wireless telecommunication, including fixed wireless access (FWA), cellular network, and satellite network. Chief of Indonesia Telecommunication Association, Ririek Adriansyah, stated that by 2020 the telecommunication industry was able to show 7% positive growth.3\n\nThe Indonesian government body targeted the telecommunication industry as one of the priority industries to be developed in the government plan of national industry development 2015-2035.4 Within the report, the telecommunication industry development roadmap was separated into three phases: developing transmission-based telecommunication (radar and satellite) and smart mobile phones. The phases stipulate the development of requisite technologies, such as telecommunication and computation device integration, and high-speed wireless and optical infrastructure. Various resources are required to support industrial development, such as natural resources, human capital, innovation capability, and creativity, which ultimately lead to competitive industrial advantage.\n\nStrategic Innovation Management refers to the whole innovative practice which involves competition mechanism analysis, such as creating an innovative vision, business strategy alignment, strategy dissemination to entire organization levels, market tendency, technology, and competitor actions. Since innovation became one of the primary resources required to develop the Indonesian telecommunication industry, this research aims to understand how strategic innovation management influences telecommunication firms’ innovation performance.\n\nThis research aimed to investigate the influence of innovation strategy, organizational structure, technological capability, and customer-supplier relationship, shown as the common practices of strategic innovation management in literature on firm innovation performance.5 Figure 1 illustrates how the variables interact as the basis of research hypotheses development.\n\nH1: Innovation strategy positively influences Firm Innovation Performance.\n\nVarious prior studies supported the first hypothesis.6,7 One prior study found that Innovation strategy positively influences Firm Innovation Performance.5 Indicating that firms implementing innovation strategy would increase their innovation performance better than those who do not.\n\nH2: Organizational structure positively influences Firm Innovation Performance\n\nOrganizational structure was the second variable of strategic innovation management studied. Akhter et al.8 support the notion that organizational structure influences organizational performance. Therefore, the second hypothesis to test in this research indicates that individuals would perform their task better and become more productive with a clear structure.\n\nH3: Innovation Culture positively influences Firm Innovation Performance\n\nNext, this study would like to examine the relationship between innovation culture with firm innovation performance. The connection was investigated in prior research that found that the organization's higher innovation perception would lead to higher firm performance.9 The same notion was used as the third hypothesis in this study.\n\nH4: Technological Capability positively influences Firm Innovation Performance\n\nThe fourth hypothesis was supported by prior research examining the influence of technological capability on firms’ financial and competitive performance.10 The research categorized a firm’s technological capability into technology intensity and technology diversity, in which case both are found to positively influence a firm’s performance.\n\nH5: Customer and Supplier Relationships positively influence Firm Innovation Performance\n\nA recent study found that supplier and customer relationships became one of the critical factors in business competition.11 Suggesting that firms should build, maintain, and engage better with their customer and supplier to excel in their industry.\n\n\nMethods\n\nThis study employed a quantitative method with descriptive purpose. The study objects were the firms in the Indonesian telecommunication industry. Analysis was done on the organizational level. The study took place in 2021 with data collection taking place between April-July.\n\nThe population of research objects in this study were the firms in the Indonesian telecommunication industry listed in Indonesian Ministry of Communication and Informatics (Kominfo). A total of 843 firms were found and categorized in Table 1.2 The sample size was determined using Equation 1. With 10% margin of error, the formula resulted in a required sample size of 90 for the research.\n\nTo ensure sample firms would be able to represent the industry subsector, a population proportional stratified random sampling technique was employed to obtain the required research samples using Equation 2. The firm categories were used as the basis for subpopulation determination. The sample size for each subpopulation is presented in Table 1. The sample firms amounted to the calculated sample size and were randomly selected out of the population list and described in Table 2.\n\nData was gathered with a questionnaire containing 43 questions adapted from a prior study.5 A copy of the questionnaire can be found under Extended data.12 The questionnaire was disseminated to sample firm representatives through company email, social media (Instagram and LinkedIn), and private message.\n\nBivariate Pearson technique were used to test the validity of the questionnaire. The questionnaire was disseminated to thirty respondents and tested with 5% degree of significance and R value of 0,361, resulting in 43 valid questions out of 47 total questions in the initial questionnaire. Consequently, the invalid question items were deleted from the questionnaire later disseminated to sample firms.\n\nDescriptive analysis and structural equation model (SEM) analysis were used to analyze the data. This was used to measure the current implementation of measured variables.9 For each variables’ indicators, the result of respondents’ answers were divided by the ideal score of the questionnaire (score obtained when the respective maximum score answered each question). Afterward, the percentage score would be compared with the given category. A score between 25-43.75% meant the indicator was very lowly implemented; a score between 43.76-62.5% meant the indicator was lowly implemented; a score between 62.6-81.25% meant the indicator was highly implemented; Lastly, a score between 81.26-100% meant the indicator was very highly implemented.\n\nTo measure the correlation between variables, in order to determine significantly influencing variables, the Partial Least Square (PLS) method was chosen as the measurement method. This provides consistent and reliable results and can be applied to complex structural equation models with many constructs.11,13 With the absence of a requirement of normally distributed input data and a high requirement on sample size and distribution compared to covariance analysis.14,15 The PLS model consists of two components: the measurement model (outer model) and the structural model (inner model). The outer model relates the observed manifest variable to the latent variable, while the inner model describes the relationship between latent variables in the SEM-PLS model.\n\nThe outer model's evaluation criterias comprise convergent validity, discriminant validity, and reliability. Convergent validity is how a measure is positively correlated with alternative measures of the same construct. High outer loadings on the constructs indicate that the related indicators have many similarities captured by the constructs.16 An acceptable value for convergent validity is if the loading factor value was higher than 0.7 and the average variance extracted (AVE) value was higher than 0.5.17 Discriminant validity is the extent to which a construct is utterly different from other constructs by empirical standards. It will be considered acceptable if the square root AVE is greater than the inter construct correlation coefficient.18 Lastly, reliability is measured through composite reliability (CR) with the criteria of CR > 0.7, which means it has high reliability.\n\nThe inner model evaluated by the value of R2 and f.2 The value of R2 was used to measure the degree of change from the independent variable to the dependent variable. A higher R2 value indicates a better predictive capability of the model. F2 shows a change in the value of R2 in the endogenous construct.12 Changes in the value of R2 indicate whether the exogenous construct has a substantive effect on the endogenous construct. If the value of f2 < 0.02, then the effect of exogenous latent variable is insubstantial, 0.02 < f2 < 0.15 is weak, 0.15 < f2 < 0.35 is moderate and f2 > 0.35 is a strong category.\n\n\nResults\n\nTable 3 summarizes the descriptive analysis of the respondents’ answers. For each studied variable, the total score obtained from the questionnaire was divided by the ideal score for the given question. They resulted in a percentage score on how each variable can achieve the desired score. The descriptive analysis result indicates that strategic innovation management has been highly implemented in Indonesia's telecommunication industry.\n\nSEM-PLS analysis tested the hypotheses developed in this research through the conceptual model depicted in Figure 2. The model consisted of the measurement model (outer model) and structural model (inner model). In the outer model, we evaluate the convergent validity, discriminant validity, and internal consistency. At the same time, the inner model was evaluated using R2 and coefficient path or T-values.\n\nThe measurement model evaluated the validity and reliability of the model developed in this study. Loading factor, internal consistency (Cronbach’s α and composite reliability/CR), and the value of average variance extracted (AVE) were the criteria used to determine the convergent validity of this research instrument. At the same time, cross-loading value and Fornell-Lacker criterion were used to determine the discriminant validity of this research instrument.\n\nThe loading factor shows the correlation between an indicator score and to construct indicator score of the given construct. An indicator should be valid as a research instrument if the loading factor value is higher than 0.7.19 The development of variables indicators was iterated several times before finding valid research indicators described in Table 4.\n\nCronbach’s α and composite reliability (CR) were used as criteria to determine the reliability of research variables. Although Cronbach’s α was a frequent measurement used,20 CR was the main criterion in this research considering CR does not assume the weight equality of each indicator. The result of Cronbach’s α and CR value of the research variables is shown in Table 5. The result indicates that the variables were internally consistent, considering the value of Cronbach’s α or CR was higher than 0.7.\n\nThe last criteria to determine the research instruments' convergent validity was the value of average variance extracted (AVE). Table 6 shows the result of the AVE test for every research variable used. The AVE value of all research variables was higher than 0.5, indicating that the research instruments fulfill the criteria of convergent validity.\n\nThe discriminant validity test of research instruments was conducted by observing the value of cross-loading of variable indicators. The cross-loading value shows the correlation of each construct from the same indicators and with the other indicators. A measurement model should be discriminantly valid if the correlation value within the same indicator is higher than the correlation value outside the indicator. The result cross-loading test of this research instrument is shown in Table 7. The bold numbers in the table mark the inter-indicator correlation. As the correlation value intra-indicator was higher than inter-indicator, these research instruments were found to fulfill the criteria to be discriminantly valid.\n\nLastly, the research instrument was tested using the Fornell-Lacker criterion to determine its discriminant validity. Table 8 shows the result of the test. The bold numbers were the square root value of AVE, while the numbers under them were the correlation value between the constructs. Because the square root value of AVE was higher than the correlation value, this research instrument should fulfill the criteria to be discriminantly valid.\n\nThe second test in evaluating the SEM-PLS model was its structural model (inner model). The structural model was evaluated by observing the value of R2 for the dependent construct and the t-statistics value from the path coefficient test. The R2 value indicates the variance of the dependent variable caused by independent variables. This research found that the R2 value of the dependent variable amounted to 0.540. It indicated that 54% of changes in firm innovation performance were caused by strategic innovation management.\n\nSEM-PLS model developed in this research was further evaluated by the f2 value to determine the degree of effect for each studied variable. Table 9 describes the result of the f2 test for each variable. The degree of effect indicated by the f2 value was categorized as follows: a value between 0.02-0.14 was weak, a value between 0.15-0.34 was moderate, and a value higher than 0.35 was strong. This research found that only CSR had a moderate effect on FIP from the four independent variables, while the others had a weak effect on FIP.\n\n\nDiscussion\n\nThis research studied the effect of strategic innovation management on firm innovation performance under five research hypotheses, representing how the implementation of strategic innovation management influences firm innovation performance. Figure 3 shows the SEM-PLS model developed in this research. Furthermore, Table 10 describes the model's interpretation in testing the hypotheses. The rules of thumb used in this research were a hypothesis rejected if its path coefficient value was higher than 1.96 and the significance level (p-value) was higher than 0.05.\n\nFrom the hypotheses test result presented in Table 10, we can conclude that innovation strategy as a research variable had no significant positive influence on firm innovation performance. Innovation strategy was the highest level of innovation practice that includes creating an innovative vision, business strategy alignment, strategy dissemination to entire organization levels, market tendency, technology, and competitor actions. Diving more into respondent characteristics, we could observe the probable cause of the test result. The absence of strategic implementation of innovation in the mid-size firms in the Indonesian telecommunication industry presumably became the source of our finding regarding the relationship between innovation strategy and firm innovation performance.\n\nNext, the test results implied that organizational structure was not found to have a positive influence on firm innovation performance. Numerous firms within the Indonesian telecommunication industry were yet to have the capability of cross-functional responsibility. It was a novel capability to ensure strategic decision making, conflict resolution, and effective coordination in the innovation process.\n\nOn the other hand, a positive influence was implied from the test results between innovation culture towards firm innovation as research variables. The main concept of innovation in culture is creativity, openness, acceptance of new ideas, risk-taking attitude, and entrepreneurial mentality. Indonesian telecommunication firms promote risk-taking behavior, appreciate success, and are open to innovation by giving their employees a certain degree of independence to experiment.\n\nThe second accepted hypothesis was the positive influence of technological capability on firm innovation performance. Technological capability is defined as a firm's ability to conduct technical and business activities, including the efficient development of new products or processes, implying that the more firms develop their technological capability, the better they perform strategic innovation. Indonesian telecommunication firms have shown an excellent technological capability to answer customers’ needs. However, some could not maximize their capability due to their size and age.\n\nInnovation drove firms to have a market-based perspective. Evaluating customers and suppliers as partners would allow firms to maximize efficiency in exploiting rare resources and developing current capabilities. Lastly, customer and supplier relationships were found to influence firm innovation performance positively. The Indonesian telecommunication industry is indicated to have shown excellent capability in managing their relationship with their customers and suppliers.\n\n\nConclusions\n\nThe implementation of strategic innovation management in the mid-size firms of the Indonesian telecommunication industry was high. In the overall response of 94 respondents it was found that they had been implementing strategic innovation management at almost 96%. The result of this research implies that the Indonesian telecommunication industry had the capability to compete strategically, by investigating their innovation strategy, organizational structure, innovation culture, technological capability, and customer and supplier relationship.\n\n\nData availability\n\nTelkom University Dataverse: The Influence of Strategic Innovation Management on Firm Innovation Performance in Indonesian Mid-Size Telecommunication Industry. https://doi.org/10.34820/FK2/CDBQPC.12\n\nThis project contains the following underlying data:\n\n- Questionnaire Design.tab (The design of the questionnaires distributed to respondents)\n\n- Questionnaire Result.tab (Questionnaire responses)\n\nThis project contains the following extended data:\n\n- Blank Research Questionnaire.pdf (The blank version of the questionnaire distributed to respondents)\n\n- Kuesioner Riset.pdf (The original Indonesian version of the questionnaire distributed to respondents)\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\nEthical approval and consent\n\nThe authors declared that this work was approved by the Dean of School of Economic and Business Telkom University with letter No. 020/PNLT1/EB-DEK/2021.Written informed consent for publication of the participants’ details and/or their images was obtained from the participants.\n\n\nAuthor contributions\n\nConceptualization, R.R., B.M., L.U.; methodology, R.R., B.M., L.U.; software, L.U.; data curation, R.R., L.U.; visualization, L.U.; validation, R.R., B.M.; analysis, R.R., B.M., L.U.; writing – original draft preparation, R.R., B.M.; writing – review & editing, R.R., B.M., L.U.; project administration, B.M.; supervision, R.R. All authors have read and agreed to the published version of the manuscript.",
"appendix": "References\n\nKementerian Perindustrian Indonesia: Analisis Perkembangan Industri Indonesia. Kementerian Perindustrian Indonesia;2020.Reference Source\n\nBadan Pusat Statistik Indonesia: Statistik Telekomunikasi Indonesia. Badan Pusat Statistik Indonesia;2019.Reference Source\n\nSetiawan K: Industri Telekomunikasi Diprediksi Tumbuh 7 Persen, Ditopang Data - Bisnis Tempo.co. Tempo.co;2020. (accessed Mar. 30, 2022).Reference Source\n\nKementerian Perindustrian Indonesia: Rencana Induk Pembangunan Industri Nasional 2015-2035. Kementerian Perindustrian Indonesia;2015.Reference Source\n\nKalay F: The impact of strategic innovation management practices on firm innovation performance. Pressacademia. Sep. 2015; 2(3): 412–412. Publisher Full Text\n\nChen Q, Wang C-H, Huang S-Z: Effects of organizational innovation and technological innovation capabilities on firm performance: evidence from firms in China’s Pearl River Delta. Asia Pac. Bus. Rev. Jan. 2020; 26(1): 72–96. Publisher Full Text\n\nKim H, Kim E: How an Open Innovation Strategy for Commercialization Affects the Firm Performance of Korean Healthcare IT SMEs. Sustainability. Jul. 2018; 10(7): 2476. Publisher Full Text\n\nAkhter N, Rafique A, Rafiq I, et al.: Organization structure and firm performance in financial development for perspective in Coca Cola beverages in Pakistan. Int. Rev. Manag. Bus. Res. 2016; 5(2): 494.\n\nAl Kabi S, Almarri K: The mediating effects of innovation culture on leadership traits, leadership style, and firm performance. Aust. J. Basic & Appl. Sci. 2019. Publisher Full Text\n\nPark JH, Chung H, Kim KH, et al.: The Impact of Technological Capability on Financial Performance in the Semiconductor Industry. Sustainability. Jan. 2021; 13(2): 489. Publisher Full Text\n\nIqbal A, Hayat M: Supplier firm and customer firm relationship on the performance of working capital management. International Journal of Finance, Insurance and Risk Management. 2020; X(3): 53–65.\n\nRismayani R: The Influence of Strategic Innovation Management on Firm Innovation Performance in Indonesian Mid-Size Telecommunication Industry. Telkom University Dataverse;2022. Publisher Full Text\n\nUrbach N, Ahlemann F: Structural equation modeling in information systems research using partial least squares. Journal of Information technology theory and application. 2010; 11(2): 5–40.\n\nWahyuningtyas R, Disastra G, Rismayani R: Toward cooperative competitiveness for community development in Economic Society 5.0. Journal of Enterprising Communities: People and Places in the Global Economy. 2022. Publisher Full Text\n\nHaryono S: Metode SEM untuk penelitian manajemen dengan AMOS 22.00, LISREL 8.80 dan Smart PLS 3.0. Jakarta:Badan Penerbit PT. Intermedia Personalia Utama;2016.\n\nHair JF Jr, Hult GTM, Ringle CM, et al.: A primer on partial least squares structural equation modeling (PLS-SEM). Sage Publications;2021.\n\nBagozzi RP, Yi Y, Phillips LW: Assessing construct validity in organizational research. Adm. Sci. Q. 1991; 36: 421–458. Publisher Full Text\n\nFornell C, Larcker DF: Evaluating structural equation models with unobservable variables and measurement error. J. Mark. Res. 1981; 18(1): 39–50. Publisher Full Text\n\nErmawati A: Pengaruh Brand Image dan Brand Trust Terhadap Purchase Decision Produk United. Agora. 2018; 6(2).\n\nSan WH, Von WY, Qureshi MI: The Impact of E-Service Quality on Customer Satisfaction in Malaysia. J. Mark. Inform. Syst. 2020; 3(1): 46–62. Publisher Full Text"
}
|
[
{
"id": "147973",
"date": "28 Nov 2022",
"name": "Ariful Islam",
"expertise": [
"Reviewer Expertise Entrepreneurship",
"Innovation",
"Strategy",
"Small Business"
],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nTry to upgrade your problem statement with more recent citations. The introduction section should include 4 to 6 latest references (2018 to 2022) from well known journals in the field and appropriate extracts from them to motivate the researchers in the subject.\n\nPut more focus on underlying theories of the study.\n\nDevelop your conclusion segment (Theoretical contributions + Practical implications + Recommendation)\n\nIs the work clearly and accurately presented and does it cite the current literature? No\n\nIs the study design appropriate and is the work technically sound? Partly\n\nAre sufficient details of methods and analysis provided to allow replication by others? Partly\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\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": "151866",
"date": "28 Nov 2022",
"name": "Antonio Abreu",
"expertise": [
"Reviewer Expertise Industrial 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 paper deals with a very interesting topic, and it included interesting ideas. In general, I appreciate the aims of this work. However, I have the following main concerns:\nThe research question is not well contextualized.\n\nThe literature background is poor and underdeveloped.\n\nThe limitations of this work deserve a deeper discussion.\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Partly\n\nAre sufficient details of methods and analysis provided to allow replication by others? Partly\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Partly\n\nAre the conclusions drawn adequately supported by the results? Yes",
"responses": []
}
] | 1
|
https://f1000research.com/articles/11-956
|
https://f1000research.com/articles/12-31/v1
|
09 Jan 23
|
{
"type": "Systematic Review",
"title": "Entrepreneurial leadership and global management of COVID-19: A bibliometric study",
"authors": [
"Andi Yusniar Mendo",
"Sanju Kumar Singh",
"Irwan Yantu",
"Raflin Hinelo",
"Agus Hakri Bokingo",
"Elvie Febriani Dungga",
"Andi Juanna",
"Ahmad Kusuma Wardhana",
"Ballav Niroula",
"Thinzar Win",
"Sanju Kumar Singh",
"Irwan Yantu",
"Raflin Hinelo",
"Agus Hakri Bokingo",
"Elvie Febriani Dungga",
"Andi Juanna",
"Ahmad Kusuma Wardhana",
"Ballav Niroula",
"Thinzar Win"
],
"abstract": "Background The coronavirus disease 2019 (COVID-19) pandemic has caused uncertainty in many economic sectors. An entrepreneurial leadership style can become an alternative method of leadership for facing uncertainty.\n\nMethods\n\nThis study uses secondary data from the Scopus website as samples. The samples are papers from Indonesia, China, and the USA. Data were collected through the Scopus website, using keywords entrepreneurial and leadership, saved into a CSV file, and processed using VOSviewer. The findings were analyzed using a systematic search.\n\nResults\n\nEntrepreneurial leadership as a topic was more prevalent in China than Indonesia and the USA. There were 101 papers from Chinese authors, 28 from Indonesian authors, and 575 from USA authors. However, there was no topic of entrepreneurial leadership connected to the strings of the topic of COVID-19. This study also found that inclusive leadership was used in China and local government leadership was used by the USA government to anticipate the impact of COVID-19.\n\nConclusions\n\nEntrepreneurial leadership was not used for COVID-19 pandemic management in USA, China, and Indonesia as a research trend.",
"keywords": [
"COVID-19",
"economic growth",
"leadership",
"Scopus",
"topic."
],
"content": "Introduction\n\nEntrepreneurial leadership is a new concept of leadership that can leverage the potential of the human resources in the company (Strubler and Redekop, 2010). Moreover, entrepreneurial leadership is said to be more suitable than other types of leadership in facing the uncertainty of the business world. An entrepreneur is a leader for themselves and their company. Leadership is the activity of persuading people to work together in achieving a goal; the desire to achieve a impactful communication that results in influencing the actions of others.\n\nBased on a study by Okudan and Rzasa, entrepreneurial leadership emerged as a unique type of leadership in response to the development of small and medium-sized enterprises (SMEs) rather than big companies (Okudan and Rzasa, 2006), because this type of leadership believed that SMEs were the most dynamic type of business in the ever-changing market trends. Moreover, SMEs have to adapt to the fast-paced, competitive business environment which was common in the fourth industrial revolution (Saunila, 2014).\n\nSince 2020, the global economy has suffered because of the coronavirus disease 2019 (COVID-19) pandemic. Many economic sectors have been affected and could not maintain their daily progress as a result of declining demand for their products. As a result, there have been waves of unemployment, company bankruptcies, and economic recessions have occurred in several countries (Nag and Puniani, 2021). The global market has recently become unstable and uncertainty surrounding the economic condition has increased with the pandemic (Filipe, 2021).\n\nLeadership has become key for companies and institutions to survive the uncertainty of changing economic conditions. Entrepreneurial leadership, which has claimed to be able to leverage the potential of each human resource, could become the solution to the uncertainty of business, such as lack of demand in retail business as a result of lockdown and physical distancing. A study by Akter stated that there was uncertainty around the rapid decline in product demand, limitations in the workspace as a result of social distancing, problems with supply chains as a result of full and partial lockdowns, as well as declining productivity as a result of disease which could infect the human resources (Akter, 2020). Maintaining the health of human resources during the COVID-19 pandemic has become the main concern of entrepreneurial leadership, because without human resources, the production process in a company will be impeded (Septiarini, Filianti, and Suprayogi, 2020).\n\nBesides human resources, research and development have also been needed to create more innovation. Innovation has been needed during the pandemic to create more opportunities for institutions, along with the creation of new products to improve their revenue (Galanou and Farrag, 2015). Research and development (R&D) produced innovations for industries, so that it could become an inseparable part of the business world. A study by Mendenhall et al. stated that R&D has an important role in increasing firm value because it is an intangible asset that has a focus on technology and science. Startups and new firms in the fourth industrial revolution have to utilize any form of digital technology to create their own market opportunity, so that they can expand their business fast. R&D investments were made to deal with high uncertainty of product demand where innovation is needed (Mendenhall et al., 2017).\n\nAs a policy maker, governments should give more support to the research and development activities in society (Naz, 2019). Moreover, during the COVID-19 pandemic, the implementation of limited physical contact and lockdown should be accompanied by research and development activities to help companies, especially SMEs, to create more opportunities that can save them (Nag and Puniani, 2021).\n\nA paper in an academic journal published by a reputable publisher, for example, Springer, is one of the results of research and development activities. International standardized publications can demonstrate many insights and innovations in any sector (Afeyan and Cooney, 2020). Government and industrial sectors could rely on the publications to gain insight to help them to create innovations for their product or service (Farrukh et al., 2021).\n\nThis study was different from Mendenhall et al.’s (2017) previous study which emphasized the importance of R&D in a business and Akter’s (2020) about the importance of leadership in growing a business and creating a strong organization. This study emphasized the leadership type used by the government of China, United States of America, and Indonesia in managing COVID-19 impact. Based on the research above, this study aims to observe if the topics related to the COVID-19 pandemic in China, USA, and Indonesia had a correlation to entrepreneurial leadership and what sub-topics lie behind these correlations.\n\n\nLiterature review\n\nPublication in a journal is a requirement for academics to build their careers (Veer, Khiste, and Deshmukh, 2018). To “publish or perish” means that if an academician doesn’t publish a paper, he/she would not be known in academic society. However, publications can also provide guidance to the industrial sector and to bureaucrats to further develop their products and services. Because publications with many publishers are a record of the latest research findings and processes, repository users can utilize publications and integrate the insight into their own research papers (Barus and Mungkasi, 2019).\n\nEach journal is ranked based on quality. One of the most well-known indexing institutions for many journals at the international level is Scopus. Scopus has grown over the last decade because of the public openness of its data (Anna, 2018). Users can search for information related to the journals and the papers that have been indexed by Scopus under Scopus Preview. But, if they want more detailed information about papers and affiliation, they can pay a subscription fee to Elsevier. Moreover, through publications, reports of research can be preserved and used for sustainable research activities (Abukhait, Bani-Melhem, and Zeffane, 2019).\n\nWhen COVID-19 hit the world, it caused disruption to many countries, and the disruption also affected the global economy. The effect of the COVID-19 pandemic has forced many governments to implement full lockdowns, partial lockdowns, and place limits on social activity in public and private spaces. The effect of limiting the movement of society, has caused the purchasing power in society to decrease too. Purchasing power in this case means the capability of society to buy products based on their economic power. Purchasing power is also the ability of the consumers to buy the goods they need and this always fluctuates based on some factors, which are the index of consumer’s belief in a product, the index of the economy’s expectation, and the condition of the real economy (Tsai, Tao, and Yuadi, 2019). People's purchasing power is closely related to demand. The index of consumer belief was how far the consumers want to consume more of certain product, for example consuming more chicken from KFC because of its tastiness. The index of economy expectation was the expectation of economic growth, both by industry and society. The condition of real economy was the current condition of the economy. Those three factors could lower or increase the purchasing power of society. The lower the purchasing power, the less people can afford to buy products at high prices and demand for products at low prices increases (Golar et al., 2020).\n\nThe decrease of purchasing power since the beginning of the pandemic has led to volatility, uncertainty, complexity, and ambiguity in the business world, known as VUCA. Volatility refers to the unpredictable changes in a society which could affect the business result. Uncertainty refers to the inability to predict the present situation and the future, because of tough competition. Complexity refers to the many factors that could disrupt the business world and they were interconnected to each other. Ambiguity refers to lack of clarity from people and organizations to make sure that the business plan could be guaranteed for expecting result (Sukoco, Suprayogi, and Hidayati, 2018) VUCA is the term used by Warren Bennis and Burt Nanus to explain an uncertain economy where it is difficult for business owners to anticipate the future during the pandemic, the business world has struggled to predict opportunities for good business. Moreover, strategies that were used effectively before the COVID-19 pandemic were not effective anymore because the situation was completely different (Johansen and Euchner, 2013).\n\nEntrepreneurial leadership could match the VUCA condition, because this type of leadership allows employees to be in charge of their own job responsibilities with minimum supervision, to give them more freedom in working. Moreover, entrepreneurial leadership emphasizes the role of employees as the core of the company and encourages them to contribute to decision-making for the company or institution, instead of leaders controlling decision-making (Renko, 2017).\n\n\nMethods\n\nThis study used secondary data in its analysis, obtained from the Scopus database. Scopus gives robust information related to publications via a paid subscription service, or Scopus Preview can be used for free although it has limited functions. Scopus meta data of all papers that have been published by journals indexed by Scopus can only be accessed through subscription. Data such as number of citations, author’s name and affiliation, name of journal, h-index of journals and papers, content of papers such as title and abstract, as well as how many journal issues are published every year, in all topics or in a specific topic. This study used purposive sampling in gathering data from Scopus.\n\nIn this study, samples were publications written by authors in China, Indonesia, and the USA. The time frame was 2020-2021 and only papers using English language were selected as samples. The studies selected were also limited to “medicine”, “business”, “management” and “social science”. The data were collected on 25 August 2021 through Scopus using “advanced search”. From “advanced search”, the query formula was entered to retrieve relevant results. The formula was “TITLE-ABS-KEY ((ENTREPENEUR*) AND (LEADERSHIP) (COVID) AND (CHINA/INDONESIA/USA))”. “TITLE-ABS-KEY” means that any topic related to “entrepreneurial leadership” and “covid” would be searched only at the sections of “title”, “abstract”, and “keywords” in any paper (Sa’ed and Al-Jabi, 2020). This study found out that there were 86 papers written by Chinese authors that covered the relevant topics, 27 by Indonesian authors, and 999 by American authors. These papers were all analyzed in this research, and none were excluded. The samples ranged from January 2020 to 25th August 2021.\n\nData were collected from papers indexed in Scopus by using keywords in search feature. Those keywords were “TITLE-ABS-KEY ((ENTREPENEUR*) AND (LEADERSHIP) AND (CHINA/INDONESIA/USA))”, by selected purposed criteria: “citation information”, “bibliographic information”, and “abstract & keywords”. These details were exported into comma separated value file format (CSV), so that the data could be analyzed by bibliometric software. The CSV file was analyzed using VOSviewer® software version 1.6.16, as this version was able to identify research trends as well as connections between the research topics and previous publications. VOSviewer was used to identify which topics were closely related to COVID-19 and leadership. VOSviewer could manifest the relationship between topics into connected circles (Mafruchati and Makuwira, 2021).\n\nA literature review was also conducted to analyze the findings of the papers that were related to the topic of this study. There were three papers from China, four from Indonesia, and two from the USA. The literature review was documented in tables including topic and findings of the papers. According to Dahlawi et al. (2021), a literature review should, at minimum, have a sample of ten papers before conducting analysis using those papers.\n\nThe comma separated file (CSV) that was downloaded from Scopus by using keywords “leadership” and “covid”, was then extracted into an Excel file (xls) using bibioshiy from R studio. The papers included were those with abstracts that discussed “leadership” and “covid” (Shi and Li, 2019). The screening results only included papers with abstracts that had results related to leadership and COVID-19 management. According to Toorajipour et al. (2021), to get the result of the research question, at least 10 papers have to be used for literature review and study used 11 papers from Chinese and Indonesian authors, and 10 from USA authors.\n\n\nResults and discussion\n\nThere were 101 papers written by Chinese authors. The characteristics of these papers were more focused on health management than leadership Figure 2 shows that the keyword “COVID-19” was connected to the keywords “inclusive leadership”. There were 101 papers which contained the keyword COVID-19, which we found by searching that keyword in the CSV file. There were three papers that stated that inclusive leadership was used by the Chinese government to manage impact of COVID-19. The keyword “COVID-19” was also connected to some keywords such as “public health”, “crisis management”, “nursing”, “collective action”, and “psychological safety”, which were related to the medical action associated with the COVID-19 pandemic. Interestingly, only the keyword “crisis management” appeared in papers from 2021, which is marked with the yellow colored dots. Moreover, as Figure 2 shows, there was no entrepreneurial topic at all that was closely related to the topic of COVID-19. Figure 2 above shows the list of keywords which appeared in papers published between 2020-2021, as well as the timeline of the keywords, represented by the colored bar in the bottom right of the image. The full dataset can be found under Underlying data.\n\nBased on Table 1, inclusive leadership could improve career sustainability. This is surprising because China had the second-highest GDP after the USA in 2020 and did not emphasize entrepreneurial leadership, but inclusive leadership during the pandemic (Ahmed et al., 2021). Table 1 also shows that the government of China should have paid more attention to public health, so that the medical action could be implemented in a quick response after the pandemic started to spread. Lack of governmental awareness toward public health would cause loss of life and would burden the government with more expenses in order to care for infected patients (Shah et al., 2020).\n\nSurprisingly, based on Table 1, people who have been able to work in the office had the self-awareness to follow health protocols. People that want to return to their workplace need the safety guaranteed by the government and institutions that the area will be free from virus dissemination, safe to work in using standardized health protocol, and assurance that their workload won’t make them sick, because sickness could make them more vulnerable to infection (Miftahussurur et al., 2022). Moreover, people want to make sure that when they return to work, their workplace isfree from any contagious disease. As a result, people prefer working from home because it is safer (Mafruchati, 2020).\n\nFigure 3 shows that there was no correlation between topics related to COVID-19 and leadership in Indonesia. There was a topic of transformational leadership, but no string was attached from this dot to dots for major topics such as “pandemic”, “Indonesia”, or “covid-19”. Research trends in 2021 focus more on the management of the pandemic or medical topics such as Indonesian and pandemic, ascaris, albendazole, World Health Organization (WHO), and epidemic. But there was a recent topic that is also connected to topics of Indonesian and pandemic which is capitalists. There was also the topic of innovation which is related to one of the key factors in entrepreneurial leadership. Other topics that may be related to leadership are employee performance and commercial phenomena.\n\nAs shown in Table 2, the topic “innovation” found that remote work behavior can increase innovation. Rizou et al. (2020) stated that digital technology could facilitate leaders to encourage innovation. Digital technology equipped with the latest artificial intelligence could lead to finding more innovation and revolutionary ideas, but this does have a high monetary cost (Ratnasari et al., 2020).\n\nAdditionally, using a virtual meeting place can help to cut costs during the pandemic, because companies can save on the operational cost for preparing and using a physical room. However, working from home for long periods of time could lead to increased stress for the employees, because employees cannot interact face-to-face with each other, which creates a lack of emotional interaction (Liao and Huang, 2016). Humans are social creatures that need to communicate physically to stabilize their mental health. Lack of social interaction could led to stress (Pei, Gunawan, and Jen, 2014). Working from home also impedes the ability to rest, because companies and institutions think that employees are available to work anytime, causing employees increased stress (Miguel-Puga et al., 2020).\n\nUnder entrepreneurial leadership, the leader of the company or institution should attempt to understand the effects of the pandemic better and try to minimize the employees’ workload accordingly. Stress from a high workload can affect the immune system’s function and lead to a sudden drop in immunity against COVID-19 (Shammi et al., 2020). A leader should prioritize the work according to the vision of the company and adjust the working system to suit the current situation (Strubler and Redekop, 2010).\n\nBut in the case of Indonesia, the implementation of health procedures and an economic plan to combat COVID-19 were not entirely successful. Indonesian authority was impeded by many capital interests revolving around the management of the COVID-19 pandemic (Megatsari et al., 2020). Rather than spending the government funds through expansionary fiscal policy to increase public health conditions and facilities, many politicians used these funds entrusted by the government for their personal interests, for example for gaining political votes (Mietzner, 2020).\n\nThe Indonesian government has benefitted from the increase in remote work and has capitalized on the essential sectors that have been vital in handling pandemics, such as increasing the cost of swabs and PCR tests, and has been weak in enacting laws against those who have engaged in covid management (Djalante et al., 2020). These sectors included those testing tools for COVID-19 and those providing social aid in the form of groceries and funds. For example the case of Juliari Batubara, the former Indonesia Minister of Social Affairs, who was arrested due to corruption regarding social aid during the COVID-19 pandemic, in what was the worst case of uncontrolled management of essential sectors during COVID-19 (Caesar and Naibaho, 2021).\n\nTable 2 also shows that the supply chain has been disrupted during the pandemic, which could affect the national economy. While remote working can substitute for face-to-face meetings, it cannot be used to resolve supply chain distributions caused by transportation. In entrepreneurial leadership, a leader should recognize that a difficult situation can become an opportunity (Renko et al., 2015). The government should have established proper regulations when anticipating the difficulties in distributing the supply chain during the pandemic. The lack of supply chains for necessities such as food materials will create price bubbling in the market, which could have detrimental effects and cause consumers to suffer (Muzakki, 2020).\n\nFigure 4 shows that there was a connection between the topic of COVID-19 and leadership. Another topic that might be related to leadership was “human” as the driver of leadership. Humans were the ones who implemented the leadership system and how good or bad the results are is based on how well humans implemented their leadership for the sake of the public’s interest (Mendenhall et al., 2017).\n\nThere was also a topic of workforce, but the size was too small and too far from COVID-19 dot, so it was less significant in correlation with topics of “leadership” or “covid”. There was also no entrepreneurial leadership related to COVID-19; in fact, there was no topic of entrepreneurial leadership at all. It can be concluded that entrepreneurial leadership has not become popular enough to be used as a topic for research related to COVID-19.\n\nTable 3 shows that based on the topic of “human”, the research explained that in order to stop the spread of COVID-19 across the USA, understanding the mobility pattern of humans is key. Leaders of local and central governments in the USA have to understand humans’ mobility patterns, which are influenced by socio-economic patterns. Effective leadership involves analyzing the current condition of society and taking action. This plays a crucial role in understanding the mobility pattern. Good leaders have to know the people they will lead in order to decide the most effective way of leading them. Otherwise, friction and disagreement would be created among society (Rachmat et al., 2018). Based on Table 3, socio-economic factors also had to be considered before implementing the lockdown. Without considering the socioeconomic factors behind the mobility patterns of society, it would be difficult to completely map the pattern. Socio–economic factors always affect humans decisions, especially those related to their primary needs (Lahcen et al., 2020).\n\nTable 3 also showed that effective communication from the local government can reduce the stress levels of health workers. A study by Esmer and Faruk stated that the goal of entrepreneurial leadership is to inspire employees with the vision that they can work together. In the COVID-19 pandemic, it is essential for both employees and their leader to share the same vision, which is to overcome the virus and reduce the number of deaths and active cases of COVID-19 in society (Hossain, 2020).\n\nFigure 4 also showed that there were other COVID-19 related keywords in several papers which were published in the early months of 2020. Those keywords were also related to leadership, which were “decision making” and “information dissemination”. Decision-making should be determined carefully by leaders (Rume and Islam, 2020) because flawed decision-making by the government can further the spread of COVID-19 and has resulted in a great number of people dying (Sharma et al., 2021). If the government does not provide a good example to society, especially to health workers in regards to implementing COVID-19 measures, the rate of positive cases will be difficult to reduce (Bender, 2020).\n\nA study by Kula et al. (2021) stated that bureaucracy has to be directed by an entrepreneurial leader so that each employee’s potential can be awakened. That leader must have the ability to strategize in difficult situations such as preventing COVID-19, as well as revitalizing the local economy. COVID-19 was a serious problem for humanity which could demolish economy growth because of the restriction to gather on the public and meet with someone to prevent virus dissemination. As a result, the supply chain of the goods would be disrupted and led to the profit loss of many companies, led to the recession. Leader of each country should has strong intuition of entrepreneurial skill to overcome this pitiful situation. He/she should not only care for the entrepreneurs of the big companies, but also SMEs (Vanderford and Marcinkowski, 2015). The COVID-19 pandemic has brought misery to many sectors in Indonesia, and the government should identify any opportunity to regain economic prosperity. Leaders also have to be prepared for the risks that may come as a result of their actions (Febriyanti, Ratnasari, and Wardhana, 2022).\n\n\nConclusion\n\nBased on the results above, there is no single relationship between entrepreneurial leadership and COVID-19 as research topics in papers indexed in Scopus. For the topic of entrepreneurial leadership, China has more updated papers about entrepreneurial leadership than Indonesia, while the USA did not have an exact topic of entrepreneurial leadership. Instead, the USA combined the topics of “leadership” and “humans”. No entrepreneurial leadership keyword was found in the published papers by US authors.\n\nThe results above also show that the topic of innovation was related to the topics of leadership and COVID-19 in Indonesia. China emphasized more on the topic of ethics, which is related to the topics of leadership and COVID-19. For the USA, they emphasized the topic of human which was related to the topics of leadership and COVID-19.\n\nResults of the literature review also showed that inclusive leadership was used by the Chinese government according to the samples. Papers written by USA authors showed that local government leadership was used for COVID-19 pandemic management. Meanwhile, no leadership topic in papers were written by Indonesian authors as samples of this study. In general, leadership was more common in topics of papers written by Chinese and American authors.\n\nThis study was limited to papers on the topic of leadership available from the Scopus website written by Chinese, Indonesian, and American authors. Moreover, the systematic search as a supplement method only explained the papers that contained leadership and covid topics in their abstract. The papers in this study’s sample that were identified using this systematic search method are also very limited in number, and further studies are needed to explore more about the excluded papers. This study also recommends that further research should include more sources such as Web of Science, PubMed, or Google Scholar to provide more potential content and to enrich findings related to entrepreneurial leadership during the COVID-19 pandemic.\n\n\nData availability\n\nZenodo: Entrepreneurial Leadership toward Global Management of COVID-19, Is it always being used during Pandemic? A Bibliometric Study. https://doi.org/10.5281/zenodo.6050768 (Mendo et al., 2022).\n\nThis project contains the following underlying data:\n\n- China keyword.png [keywords of papers written by Chinese authors and correlation between them after analyzed using Vosviewer]\n\n- China COVID Topics.csv [topics of papers written by Chinese authors and correlation between them after analyzed using Vosviewer]\n\n- Indonesia COVID Topics.csv [topics of papers written by Indonesian authors and correlation between them after analyzed using Vosviewer]\n\n- Indonesia keywords.png [keywords of papers written by Indonesian authors and correlation between them after analyzed using Vosviewer]\n\n- USA COVID Topics.csv [topics of papers written by USA authors and correlation between them after analyzed using Vosviewer]\n\n- USA keywords.png [keywords of papers written by USA authors and correlation between them after analyzed using Vosviewer]\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).",
"appendix": "References\n\nAbukhait RM, Bani-Melhem S, Zeffane R: Empowerment, Knowledge Sharing and Innovative Behaviours: Exploring Gender Differences. Int. J. Innov. Manag. 2019; 23(01): 1950006. World Scientific. Publisher Full Text\n\nAfeyan NB, Cooney CL: Professor Daniel IC Wang: A Legacy of Education, Innovation, Publication, and Leadership. Biotechnol. Bioeng. 2020; 117(12): 3615–3627. Wiley-Blackwell. PubMed Abstract | Publisher Full Text\n\nAhmed F, Zhao F, Faraz NA, et al.: How Inclusive Leadership Paves Way for Psychological Well-being of Employees during Trauma and Crisis: A Three-wave Longitudinal Mediation Study. J. Adv. Nurs. 2021; 77(2): 819–831. Wiley Online Library. PubMed Abstract | Publisher Full Text\n\nAkter S: Covid-19 and Bangladesh: Threat of Unemployment in the Economy. 5. International EMI Entrepreneurship and Social Sciences Congress PROCEEDINGS E-BOOK. 2020; 280.\n\nAnna NEV: Transformation of Public Library Websites in Indonesia. 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International Journal of Applied Business and International Management (IJABIM). 2021; 6(1): 110–122. Publisher Full Text\n\nLahcen B, Brusselaers J, Vrancken K, et al.: Green Recovery Policies for the COVID-19 Crisis: Modelling the Impact on the Economy and Greenhouse Gas Emissions. Environ. Resour. Econ. 2020; 76(4): 731–750. Springer. PubMed Abstract | Publisher Full Text\n\nLiao K-H, Huang I-S: Impact of Vision, Strategy, and Human Resource on Nonprofit Organization Service Performance. Procedia Soc. Behav. Sci. 2016; 224: 20–27. Elsevier. Publisher Full Text\n\nMafruchati M: Curcumin vs Chloroquine in Coronavirus Global Pandemic: Trend Analysis Study in Google. Indian J. Forensic Med. Toxicol. 2020; 14(4): 3138–3143.\n\nMafruchati M, Makuwira J: Number of Research Papers about Agricultural Production, Meat, and Egg During COVID-19 Pandemic: Does It Changed than Before? Pharm. J. 2021; 13(4): 995–998. Publisher Full Text\n\nMegatsari H, Laksono AD, Ibad M, et al.: The Community Psychosocial Burden during the COVID-19 Pandemic in Indonesia. Heliyon. 2020; 6(10): e05136. Elsevier. PubMed Abstract | Publisher Full Text\n\nMendenhall ME, Weber TJ, Arnardottir AA, et al.:Developing Global Leadership Competencies: A Process Model. Advances in Global Leadership. Emerald Publishing Limited;2017.\n\nMendo AY, Singh SK, Hinelo R, et al.: Entrepreneurial Leadership toward Global Management of COVID-19, Is it always being used during Pandemic? A Bibliometric Study. Zenodo. 2022. Publisher Full Text\n\nMietzner M: Populist Anti-Scientism, Religious Polarisation, and Institutionalised Corruption: How Indonesia’s Democratic Decline Shaped Its COVID-19 Response. J. Curr. Southeast Asian Aff. 2020; 39(2): 227–249. SAGE Publications Sage UK: London, England. Publisher Full Text\n\nMiftahussurur M, Savitri CMA, Waskito LA, et al.: Lessons from Indonesia, a Country with Highest COVID-19 Mortality Rate in the World: Dissecting Multiple Aspects. F1000Res. 2022; 11(920): 920. F1000 Research Limited. Publisher Full Text\n\nMiguel-Puga JA, Cooper-Bribiesca D, Avelar-Garnica FJ, et al.: Burnout, Depersonalization, and Anxiety Contribute to Post-traumatic Stress in Frontline Health Workers at COVID-19 Patient Care, a Follow-up Study. Brain Behav. 2020; e02007. Wiley Online Library.\n\nMuzakki F: The Global Political Economy Impact of Covid-19 and The Implication to Indonesia. Journal of Social Political Sciences. 2020; 1(2): 76–92.\n\nNag B, Puniani A: Post-Covid World Economy and India-China Bilateral Trade. Trade Dev. Rev. 2021; 13(2).\n\nNaz S: Impact of Globalization on Higher Education in Pakistan: Challenges and Opportunities. International Journal of Innovation in Teaching and Learning (IJITL). 2019; 2(2).\n\nOkudan GE, Rzasa SE: A Project-Based Approach to Entrepreneurial Leadership Education. Technovation. 2006; 26(2): 195–210. Elsevier. Publisher Full Text\n\nPei Y, Gunawan S, Jen SC: Correlations between Social Engagement and Quality of Life of the Elderly in China. Rev. Int. Sociol. 2014; 72(2): 105–118. Instituto de Estudios Sociales Avanzados. Publisher Full Text\n\nRachmat F, Prasetya TAE, Ardyanto D, et al.: Leadership Style Correlation with the Occurrence of Unsafe Act Fabrication Employees Pt. BSB Gresik. Indian J. Public Health Res. Dev. 2018; 9(6): 23–27. Prof.(Dr) RK Sharma. Publisher Full Text\n\nRatnasari RT, Gunawan S, Mawardi I, et al.: Emotional Experience on Behavioral Intention for Halal Tourism. J. Islam. Mark. 2020; 12(4): 864–881. Emerald Publishing Limited. Publisher Full Text\n\nRenko M:Entrepreneurial Leadership. Forthcoming in “Nature of Leadership”. Day DV, Antonakis J, editors.3rd Ed.SAGE Publications;2017.\n\nRenko M, El Tarabishy A, Carsrud AL, et al.: Understanding and Measuring Entrepreneurial Leadership Style. J. Small Bus. Manag. 2015; 53(1): 54–74. Taylor & Francis. Publisher Full Text\n\nRizou M, Galanakis IM, Aldawoud TMS, et al.: Safety of Foods, Food Supply Chain and Environment within the COVID-19 Pandemic. Trends Food Sci. Technol. 2020; 102: 293–299. Elsevier. PubMed Abstract | Publisher Full Text\n\nRume T, Didar-Ul Islam SM: Environmental Effects of COVID-19 Pandemic and Potential Strategies of Sustainability. Heliyon. 2020; 6: e04965. Elsevier. PubMed Abstract | Publisher Full Text\n\nSa’ed HZ, Al-Jabi SW: Mapping the Situation of Research on Coronavirus Disease-19 (COVID-19): A Preliminary Bibliometric Analysis during the Early Stage of the Outbreak. BMC Infect. Dis. 2020; 20(1): 1–8. Springer. PubMed Abstract | Publisher Full Text\n\nSaunila M: Innovation Capability for SME Success: Perspectives of Financial and Operational Performance. J. Adv. Manag. Res. 2014; 11(2): 163–175. Emerald Group Publishing Limited. Publisher Full Text\n\nSeptiarini DF, Filianti D, Suprayogi N: IMPACT OF DISRUPTION ERA ON ORGANIZATION PERFORMANCE SUSTAINABILITY: A CASE STUDY. J. Secur. Sustain. Issues. 2020; 9. Publisher Full Text\n\nShah K, Chaudhari G, Kamrai D, et al.: How Essential Is to Focus on Physician’s Health and Burnout in Coronavirus (COVID-19) Pandemic? Cureus. 2020; 12 (4). Cureus Inc. Publisher Full Text\n\nShammi M, Bodrud-Doza M, Abu Reza M, et al.: COVID-19 Pandemic, Socioeconomic Crisis and Human Stress in Resource-Limited Settings: A Case from Bangladesh. Heliyon. 2020; 6(5): e04063. Elsevier. PubMed Abstract | Publisher Full Text\n\nSharma GD, Tiwari AK, Jain M, et al.: Unconditional and Conditional Analysis between Covid-19 Cases, Temperature, Exchange Rate and Stock Markets Using Wavelet Coherence and Wavelet Partial Coherence Approaches. Heliyon. 2021; 7(2): e06181. Elsevier. PubMed Abstract | Publisher Full Text\n\nShi Y, Li X: A Bibliometric Study on Intelligent Techniques of Bankruptcy Prediction for Corporate Firms. Heliyon. 2019; 5(12): e02997. Elsevier. PubMed Abstract | Publisher Full Text\n\nStrubler DC, Redekop BW: Entrepreneurial Human Resource Leadership: A Conversation with Dwight Carlson. Hum. Resour. Manag. 2010; 49(4): 793–804. Wiley Online Library. Publisher Full Text\n\nSukoco BM, Suprayogi N, Hidayati NA: The Effects of Market Orientation on Environmental Social Responsibility Programmes: The Moderating Effects of Institutional Pressures. Pertanika J. Soc. Sci. Humanit. 2018; 26(18): 185–202.\n\nToorajipour R, Sohrabpour V, Nazarpour A, et al.: Artificial Intelligence in Supply Chain Management: A Systematic Literature Review. J. Bus. Res. 2021; 122: 502–517. Elsevier. Publisher Full Text\n\nTsai M-J, Tao Y-H, Yuadi I: Deep Learning for Printed Document Source Identification. Signal Process. Image Commun. 2019; 70: 184–198. Elsevier. Publisher Full Text\n\nVanderford NL, Marcinkowski E: A Case Study of the Impediments to the Commercialization of Research at the University of Kentucky. F1000Res. 2015; 4: 133. Faculty of 1000 Ltd. PubMed Abstract | Publisher Full Text\n\nVeer DK, Khiste GP, Deshmukh RK: Publication Productivity of ‘Information Literacy’in Scopus during 2007 to 2016. Asian Journal of Research in Social Sciences and Humanities. 2018; 8(2): 171–183. Asian Research Consortium. Publisher Full Text"
}
|
[
{
"id": "168969",
"date": "27 Apr 2023",
"name": "Stamatios Papadakis",
"expertise": [
"Reviewer Expertise Leadership"
],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 article, and I enjoyed reading it.\nSome minor recommendations are the following:\nThe authors should please state more clearly how their approach relates to existing policies and how it advances the field in assessing citation rates and bibliometrics, in general, how their approach promotes state of art, and how relevant this is in general, and the data in particular.\nAlso, it would be very useful if the authors would make their data and source code available as supplementary material. This would promote the usage of the proposed research, allow others to take better advantage of it, and enable them to reproduce the results.\nThe conclusion does well to summarise the article’s contents; however, a short explanation of the limitations of this study is needed.\n\nAre the rationale for, and objectives of, the Systematic Review clearly stated? Yes\n\nAre sufficient details of the methods and analysis provided to allow replication by others? Yes\n\nIs the statistical analysis and its interpretation appropriate? Yes\n\nAre the conclusions drawn adequately supported by the results presented in the review? Yes",
"responses": []
},
{
"id": "171465",
"date": "23 May 2023",
"name": "Lutfi Lutfi",
"expertise": [
"Reviewer Expertise Capital market",
"Corporate Governance",
"Bank Management",
"Behavioral Finance",
"Wealth 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\nTitle\nIt would be strange if the topic of the article was about \"entrepreneurial leadership\", but research results show that \"entrepreneurial leadership\" is not used to manage Covid-19. It is better if the title is changed, for example: Does entrepreneurial leadership used for global management of COVID-19?: A bibliometric study\nKeyword\nGiven that \"economic growth\" is not the main aspect that is examined in this article, it is better if the term is not included in the \"keyword\"\nIntroduction\nThe author needs to explain the difference between this article (a bibliometric study on entrepreneurial leadership) and previous articles that also conduct a bibliometric study on entrepreneurial leadership (eg. Bauwens, R., Batistič, S., Kilroy, S., & Nijs, S. (2022). New kids on the block? A bibliometric analysis of emerging COVID-19—trends in leadership research. Journal of Leadership & Organizational Studies, 29(2), 224-232.; Aparisi-Torrijo, S., & Ribes-Giner, G. (2022). Entrepreneurial leadership factors: a bibliometric analysis for the 2000-2020 period Cuadernos de Gestión, 22(2), 45-60.; Roeschke, A. (2016). Entrepreneurial Leadership: A Bibliometric Analysis.1, p. 17614). Briarcliff Manor, NY 10510: Academy of Management., etc.). This can reveal the novelty of this article\nLiterature Review\nThe subsection \"Entrepreneurial leadership during COVID-19\" should explain why entrepreneurial leadership is so necessary for companies to survive in the face of Covid-19. However, this section discusses too much the negative impact of the Covid-19 pandemic on people's income and purchasing power. Discussion on the importance of entrepreneurial leadership for companies in conditions of uncertainty (VUCA) should be elaborated, supported by adequate recent literature\nMethods\nAuthors need to provide additional explanations why choosing articles written by authors from China, Indonesia, and the USA. Why are authors from these three countries important in the context of entrepreneurial leadership and Covid-19? Are the three countries able to represent the global management referred to in this study?\nThe title of the article mentions the term \"global management\" but in the search keywords on \"Tittle-Abs-Key\" the term does not exist. The author needs to explain this.\nAuthors need to explain how to select a sample of articles reviewed from a bibliographical sample. What sampling technique was used?\nResults and Discussion\nDiscussion of the findings should emphasize why this type of leadership is used in each country. What are the benefits of this type of leadership for the company, employees, community, or country?\nDiscussion regarding public awareness regarding health protocols, online meetings, etc. needs to be done only if it is related to the type of leadership.\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? Partly\n\nAre the conclusions drawn adequately supported by the results presented in the review? Yes",
"responses": []
}
] | 1
|
https://f1000research.com/articles/12-31
|
https://f1000research.com/articles/12-28/v1
|
09 Jan 23
|
{
"type": "Brief Report",
"title": "Validating skin swabbing as a refined technique to collect DNA from small-bodied fish species",
"authors": [
"Ceinwen Tilley",
"Lynne Sneddon",
"Eamonn Mallon",
"Iain Barber",
"William Norton",
"Ceinwen Tilley",
"Lynne Sneddon",
"Eamonn Mallon",
"Iain Barber"
],
"abstract": "DNA samples are often used to identify fish before they are utilised in other experiments. Our recent research has shown that skin swabbing can be used to collect DNA for genotyping, and that swabbing causes less harm to fish than fin clipping, another common technique. In this study we investigated potential refinements to the skin swabbing protocol by pre-treating fish with the analgesic lidocaine. We could not detect any differences in cortisol release, behaviour or expression of stress axis marker genes in skin swabbed sticklebacks or zebrafish regardless of lidocaine application. In contrast, fin clipping caused changes in cortisol release, gene expression and behaviour when analgesia was not used. These changes were rescued by pre-treatment with lidocaine confirming that analgesia was effective. The results demonstrates that skin swabbing is a refined technique for DNA collection that does not require analgesia.",
"keywords": [
"Fin clipping",
"skin swabbing",
"lidocaine",
"analgesia",
"zebrafish",
"stickleback"
],
"content": "\n\n\n\nScientific benefit(s)\n\n• Skin swabbing provides DNA of suitable quality to genotype small laboratory fish species.\n\n3Rs benefit(s)\n\n• Skin swabbing is a refined technique to collect DNA compared to fin clipping, causing fewer health and welfare changes.\n\n• It may be possible to use fewer animals in subsequent experiments when collecting DNA by skin swabbing rather than fin clipping.\n\nPractical benefit(s)\n\n• Skin swabbing is quick to perform and gives reliable results across different institutions.\n\n• There is no need to use anaesthetic, analgesic, or sharp blades when skin swabbing.\n\nCurrent applications\n\n• The current applications of skin swabbing are to collect DNA samples from small-bodied fish species for use in PCR amplification or genotyping.\n\nPotential applications\n\n• Skin swabbing might also be suitable to assay mucus samples for small metabolites or hormone levels, such as cortisol.\n\n\nIntroduction\n\nRefining the experimental techniques applied to laboratory animals is a vital part of scientific research. Experimental fish are often identified by collecting a small sample of DNA that can be used for genotyping. There are several procedures that can be used to collect DNA from fish including fin clipping and skin swabbing. Fin clipping is the more common technique, and it involves anaesthetising a fish and removing a small piece of fin tissue before allowing it to recover in fresh system water. However, there is evidence showing that fin clipping is harmful for fish, indicated by stress axis activation, changes in behaviour and negative effects on survival and growth.1–6\n\nAs an alternative to fin clipping, our recent research has investigated the welfare benefits of using skin swabbing to collect mucus samples from small fish species.7–9 The swabbing procedure involves restraining a non-anaesthetised fish upon a wetted sponge. A mucus sample is collected from the fish’s flank using a rayon-tipped swab. Skin swabbing has been shown to be less invasive than fin clipping since it activates fewer endocrine, genetic and behavioural indicators of stress and leads to less variability in post-hoc data collection.7,8 In addition, the skin swabbing protocol is easy to perform and leads to reliable DNA sampling.8\n\nIn this study we have investigated ways to improve the skin swabbing protocol. We first independently verified our study comparing changes to cortisol release and behaviour following fin clipping or skin swabbing using fish held at the University of Gothenburg. In addition, we examined whether swabbing could be improved by applying the analgesic lidocaine. Previous research suggests that analgesia administration post-fin clipping improves fish welfare.3,4 We found no difference in the impact of swabbing when carried out with or without analgesia. However, although lidocaine significantly improved the welfare of fin-clipped fish, this procedure brings additional steps to the fin clipping protocol, and lidocaine can affect fish for significant periods of time. Our results confirm that swabbing is a refined technique to collect DNA from small fish species.\n\n\nMethods\n\nAll work was conducted under a UK Home Office licence (University of Leicester, no. P8F9CCE8B) or a local ethics permit (University of Gothenburg, no. 5.8.18-16941/2021). Zebrafish and sticklebacks were kept under standard conditions in accordance with institutional guidelines for animal welfare. Three-spined sticklebacks were generated by in vitro fertilisation as described in.10 They had an average length of 39.02 ± 5.81 mm and an average weight of 0.83 ± 0.41 g. The water parameters were pH ~7.1, 0 ppm ammonia, 0 ppm nitrate, ~4 ppm nitrite and ~4000 μs/cm conductivity. AB wild-type zebrafish kept at the University of Leicester were generated by in-crossing parental stock. Zebrafish used in the lidocaine experiment had an average length of 29.67 ± 2.38 mm and an average weight of 0.24 g ± 0.15 g. Those used in the water supplements experiment had an average length of 41.13 ± 3.79 mm and an average weight of 0.94 ± 0.52 g. The water parameters were pH ~7.1, 0 ppm ammonia, 4 ppm nitrate, ~0 ppm nitrite and ~525 μs/cm conductivity. AB wild-type zebrafish kept at the University of Gothenburg were obtained from the Zebrafish Core Facility, Karolinska Institute, Stockholm, Sweden. They had an average length of 25.86 ± 1.02 mm and an average weight of 0.22 g ± 0.12 g. The water quality parameters were pH ~7.4, <0.1 ppm ammonia, <10 ppm nitrate, and <0.1 ppm nitrite. No fish of either species died during these experiments and all animals were killed by a Schedule 1 procedure at the end of the study.\n\nExperiment 1. Cortisol and measurements of behaviour (University of Gothenburg)\n\nCortisol release and measurements of behaviour using the Fish Behavioural Index (FBI) were carried out in Gothenburg. A total number of 48 zebrafish were used. Experiments were conducted as outlined in.3 Zebrafish were randomly selected and transferred to a semi-closed recirculation system containing two parallel rows of glass tanks (20 × 30 × 20 cm; n = 1 fish per tank). Fish were acclimatised in their individual tanks for two weeks prior to experimentation. They were in visual contact with adjacent tanks until the evening prior to experimentation when two opaque pieces of plastic were placed between tanks to visually isolate individuals. Fish were randomly assigned by haphazard netting from a home tank into one of the following groups: Fin Clip; Fin Clip plus Lidocaine; Skin Swab or Skin Swab plus Lidocaine (n = 12 per group). At the start of the experiment the water flow to the experimental tanks was switched off and 5 mg/L lidocaine was administered to the tank. Behaviour was compared at pre-treatment and 1, 2, 3 and 6 h afterwards. At each time point behaviour was recorded for 25 min using video cameras linked to FBI tracking software. The FBI assigns one value based upon activity and distance travelled to classify behaviour as: Healthy (0.84-1.0); OK (0.67-0.83); Unhealthy (0.33-0.66); or Abnormal (0-0.32) (Deakin et al., 2019). After the video recording, a water sample was obtained for cortisol analysis.1 Water samples were collected into two clean 120 mL containers per treatment group using siphons. Blank water samples (n = 2) were taken directly from empty tanks at each sampling point to account for residual cortisol levels (ranging from 37-56 pg/cartridge) and blank values were subtracted from the corresponding experimental sample.11 Following collection, samples were immediately frozen at -20°C and were allowed to thaw at 4°C for 24 hours before cortisol extraction at a later date. Water samples were filtered with 0.45 micron nitrocellulose filters (10 cm diameter, Whatman, UK) before undergoing solid phase extraction using C-18 Sep-pak cartridges fitted to a mini-pulse machine set at 25 ml min-1. Cartridges were first primed with 5 ml methanol and washed with 5 ml distilled water. The water sample was then passed through the cartridge, followed by a final wash with 5 ml distilled water. All cartridges were labelled and allowed to dry before being frozen for future analysis (-20°C). Cortisol was assayed blind using 200 μL of eluate in a radioimmunoassay.12\n\nExperiment 2. Administration of lidocaine (University of Leicester)\n\nIn separate experiments carried out in Leicester, we investigated the welfare impact of administering analgesia prior to DNA sampling and stress relief water supplements post-sampling. Two weeks prior to experimentation, fish were caught and were distributed by blind randomization into ten tanks on the system, (six for analgesia studies and four for water supplements with nine sticklebacks in 13.4 L tanks and nine zebrafish in 3.5 L tanks). The tanks were located in central locations of large racks within the respective species rooms, ensuring that all tanks received similar illumination and were surrounded by other tanks on the sides. No enrichment was provided. The number of fish per group was based on numbers needed for qPCR analysis. On the day of the experiment tanks were removed from the rack and placed on a trolley for 30 minutes before the start. Lidocaine studies were carried out on one day and water supplements on the following day. This was repeated two more times each week to give three technical replicates for each study. On the day of the experiment a 400 mg/L stock solution of lidocaine (Sigma-Aldrich) was prepared and diluted to 2 mg/L4 using reverse osmosis water. Lidocaine was applied by immersion for 45 min before manipulation. Six groups were investigated in both species: Non-manipulated with no pain relief (un-manipulated); Netted, swabbed and released with no pain relief; netted, anaesthetised, fin clipped and allowed to recover with no pain relief; Un-manipulated after immersion in lidocaine; Netted, swabbed and then released after immersion in lidocaine; netted, anaesthetised and fin clipped after immersion in lidocaine. The total number of individuals used were 162 sticklebacks and 162 zebrafish (9 × 6 groups × 3 independent replicates).\n\nFor details of DNA collection by skin swabbing or fin clipping, and gene expression analysis refer to.8 The expression of five stress marker genes were investigated: brain-derived neurotrophic factor (bdnf), corticotropin releasing hormone a (crha), corticotropin releasing hormone b (crhb), galanin (galn) and neuropeptide y (npy).8 The data were normalised against the geometric means of ribosomal protein L8 (rpL8), ribosomal protein L13A (rpL13A) and ubiquitin (ubiq) genes in sticklebacks and ribosomal protein L13A (rpL13A) and elongation factor 1a (elf1a) genes in zebrafish. The fold change was calculated using the 2-ddCT method.13 Primer details are shown in Table 1.\n\nPost-hoc Pairwise comparisons of lidocaine treatment and DNA method for each fish type. Degrees of freedom are 175 except for galn where they are 94 as this was not tested using zebrafish samples. 162 sticklebacks and 162 zebrafish (9 x 6 groups x 3 independent replicates) were used and 3 technical replicates were performed in the qPCR.\n\nStatistical analyses were carried out using GraphPad Prism7 and RStudio. Data were tested for normality using the using the Shapiro-Wilk test, followed by a non-parametric Kruskal-Wallis test and Dunn's multiple comparisons test comparing each treatment to the control group. Gene expression data was Log2 transformed. An ANOVA was carried out for each gene separately (model: log2(expression) = lidocaine + treatment + fish + lidocaine:treatment:fish). Post-hoc tests were carried out using the Emmeans package version 1.5.4 in R.14\n\n\nResults and discussion\n\nOur previous research has shown that skin swabbing is a refined technique to collect DNA compared to fin clipping.8 In the first set of experiments we treated 48 zebrafish with the analgesic lidocaine before either fin clipping or skin swabbing. We investigated changes to locomotion and space usage in the tank using the Fish Behavioural Index (FBI), an automated system that gives an unbiased readout of fish welfare.5 Fin clipping caused a sharp decrease in fish welfare, demonstrated by a reduction of FBI units that persisted over time (>6 h). Fin-clipped fish displayed a score of 0.18 FBI units (representing abnormal behaviour) six hours after fin clipping. Pre-treatment with lidocaine prevented this decrease in welfare. However, since FBI scores had not normalised after 6 h, lidocaine may only represent temporary relief for fin-clipped fish. In comparison, skin swabbed animals displayed similar profiles regardless of the treatment group (with or without lidocaine). All swabbed fish displayed ‘healthy’ behaviour profiles (FBI scores 0.90-0.96) (Figure 1B). We next compared excretion of cortisol. In keeping with other published studies,4,8 fin clipping in the absence of lidocaine led to heightened cortisol release compared to fin clipping with lidocaine, or skin swabbing with or without lidocaine. Importantly, there was no significant difference between the amount of cortisol excreted by zebrafish following skin swabbing with or without lidocaine, suggesting that skin swabbing is not as stressful to fish as fin clipping and that it is not refined by analgesia administration (Figure 1A). We were thus able to confirm the findings of our previous research8 using fish housed in a different country and data collected by researchers that were not involved in earlier studies.\n\nA. There is no significant difference in cortisol release between fin clipping with lidocaine treatment, or skin swabbing with and without lidocaine treatment. Treatment groups: fin clipping without lidocaine (Clip no lido); fin clipping following lidocaine pre-treatment (Clip lido); skin swabbing without lidocaine (Swab no lido and skin swabbing following lidocaine pre-treatment (Swab lido). One-way ANOVA followed by Dunnett’s multiple comparisons test. Clip no lido vs Clip lido, n=12 DF=44, p < 0.001; Clip no lido vs Swab no lido, n=12, DF=44, p < 0.001; Clip no lido vs Swab lido, n=12, DF=44, p < 0.001. B. Fin clipping in the absence of lidocaine decreases zebrafish health status compared to skin swabbing with or without lidocaine treatment. Treatment groups: fin clipping without lidocaine; fin clipping following lidocaine pre-treatment; skin swabbing without lidocaine and skin swabbing following lidocaine pre-treatment. Scores from the FBI are interpreted as follows: Healthy 0.84-1.0, OK 0.67-0.83, Unhealthy 0.33-0.66 and Abnormal 0-0.32.\n\nWe next used quantitative PCR to compare the expression level of five genes related to the stress response in sticklebacks and zebrafish: brain derived neurotrophic factor (bdnf); corticotropin releasing hormone a (crha); corticotropin releasing hormone b (crhb); galanin (gln) and neuropeptide y (npy). We compared the effect of lidocaine treatment in fish that were either un-manipulated, fin clipped or skin swabbed either with or without lidocaine (n = 27 for each treatment group, 162 in total for each species). Heightened expression of these genes would indicate higher stress levels in fish. In the absence of lidocaine, fin clipping led to heightened expression of crha, galn and npy compared to un-manipulated sticklebacks (Figure 2). There was also a significant difference in bdnf expression between skin swabbed and fin clipped sticklebacks (Figure 2). Pre-experimental lidocaine treatment heightened npy expression in sticklebacks that were not used for DNA collection, suggesting that lidocaine application can be harmful, or that the concentration and treatment regime needs to be optimised (Figure 2). However, lidocaine treatment decreased the expression of bdnf, crha and galn in fin clipped sticklebacks compared to sticklebacks that had been fin clipped without analgesia (Figure 2). There were no significant differences in gene expression between skin swabbed sticklebacks with or without lidocaine application.\n\nqPCR data showing expression relative to the geometric mean of rpL8, rpL13A and ubiq of (A) stickleback brain-derived neurotrophic factor (bdnf). (B) stickleback corticotropin releasing hormone a (crha). (C) stickleback corticotropin releasing hormone b (crhb). (D) stickleback galanin (galn). (E) stickleback neuropeptide y (npy). Groups include: 1) un-manipulated control fish (control no lidocaine); 2) skin swabbed fish (swab no lidocaine); 3) Fin clipped fish; (clip no lidocaine); 4) un-manipulated fish pre-treated with lidocaine (control lidocaine); 5) lidocaine pre-treated skin swabbed fish (swab lidocaine) and 6) lidocaine pre-treated fin clipped fish (clip lidocaine). For each group n = 27 (162 in total). Each qPCR consisted of three technical replicates. Comparisons include: a) un-manipulated no lidocaine versus fin clip no lidocaine fish (1 versus 3); b) fin clip no lidocaine versus skin swab no lidocaine (3 versus 2); c) un-manipulated no lidocaine versus un-manipulated lidocaine (1 versus 4); d) fin clip no lidocaine versus fin clip with lidocaine (3 versus 6); e) un-manipulated no lidocaine versus swab no lidocaine (1 versus 2) and f) skin swab no lidocaine vs swab with lidocaine (2 versus 5). Primer sequences are included in Table 1 and full statistical information is included in Table 2. Group legend: C = un-manipulated fish, SS = skin swabbed fish, FC = fin clipped fish.\n\nIn the absence of lidocaine there was a significant increase in npy expression in the fin clipped zebrafish compared to the skin swabbed group (Figure 3). Application of lidocaine to un-manipulated zebrafish caused a significant decrease in the expression of crha, crhb and npy (Figure 3). These findings are difficult to interpret because it is not clear what a decrease in gene expression represents in terms of fish welfare. Further research comparing gene expression to other welfare readouts is needed to clarify this point. Similarly, lidocaine treatment decreased the expression of bdnf, crha, crhb and npy in fin clipped zebrafish compared to the no-lidocaine group. Finally, lidocaine treatment also decreased bdnf and crhb expression following skin swabbing compared to the swabbing without lidocaine (Figure 3). In summary, skin swabbing is a refined technique to collect DNA, even without lidocaine application.\n\nqPCR data showing expression relative to the geometric mean of rpL13A and elf1A of (A) zebrafish brain-derived neurotrophic factor (bdnf). (B) zebrafish corticotropin releasing hormone a (crha). (C) zebrafish corticotropin releasing hormone b (crhb). (D) zebrafish neuropeptide y (npy). Groups include: 1) un-manipulated control fish (control no lidocaine); 2) skin swabbed fish (swab no lidocaine); 3) Fin clipped fish; (clip no lidocaine); 4) un-manipulated fish pre-treated with lidocaine (control lidocaine); 5) lidocaine pre-treated skin swabbed fish (swab lidocaine) and 6) lidocaine pre-treated fin clipped fish (clip lidocaine). For each group n = 27 (162 in total). Each qPCR consisted of 3 technical replicates. Comparisons include: a) un-manipulated no lidocaine vs fin clip no lidocaine fish (1 versus 3); b) fin clip no lidocaine versus skin swab no lidocaine (3 versus 2); c) un-manipulated no lidocaine versus un-manipulated lidocaine (1 versus 4); d) fin clip no lidocaine versus fin clip with lidocaine (3 versus 6). e) un-manipulated no lidocaine versus swab no lidocaine (1 versus 2) and f) skin swab no lidocaine versus swab with lidocaine (2 versus 5). Primer sequences are included in Table 1 and full statistical information is included in Table 2. Group legend: C = un-manipulated fish, SS = skin swabbed fish, FC = fin clipped fish.\n\n\nSummary\n\nWe have replicated our findings from an independent study in another facility, using different fish, equipment and researchers, demonstrating that swabbing is a refinement from fin clipping. We also confirmed that lidocaine is an effective analgesic for diminishing pain caused by the fin clipping procedure. Therefore, it is recommended that lidocaine should be provided when fin clipping is used. However, the changes in gene expression in fish exposed to lidocaine with no additional manipulation show that administration of analgesia is a procedure that can result in complex responses, including potential aversion. In addition, it is possible that longer-term welfare impacts remain once the lidocaine wears off, meaning that lidocaine treatment is not ideal. Swabbing is not further refined by administering lidocaine, suggesting swabbing does not cause discomfort. As swabbing is a refinement even in the absence of analgesia, the use of controlled substances can be avoided. We have therefore demonstrated that swabbing represents a true refinement to fin clipping for DNA collection.",
"appendix": "Data availability\n\nOpen Science Framework: Lidocaine and water conditioners, https://doi.org/10.17605/OSF.IO/6MD5V. 15\n\nThis project contains the following underlying data:\n\n- Tilley at al raw data.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\nFigshare: ARIVE Essential 10 guidelines for “Validating skin swabbing as a refined technique to collect DNA from small-bodied fish species”, https://doi.org/10.6084/m9.figshare.21603024.v1. 16\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).\n\n\nReferences\n\nWhite LJ, Thomson JS, Pounder KC, et al.: The impact of social context on behaviour and the recovery from welfare challenges in zebrafish. Danio rerio. Anim. Behav. 2017; 132: 189–199. Publisher Full Text\n\nTaslima K, Davie A, McAndrew BJ, et al.: DNA sampling from mucus in the Nile tilapia, Oreochromis niloticus: minimally invasive sampling for aquaculture-related genetics research. Aquac. Res. 2015; 47(12): 4032–4037.\n\nDeakin AG, Spencer JW, Cossins AR, et al.: Welfare challenges influence the complexity of movement: fractal analysis of behaviour in zebrafish. Fishes. 2019; 4: 8. Publisher Full Text\n\nSchroeder PG, Sneddon LU: Exploring the efficacy of immersion analgesics in zebrafish using an integrative approach. Appl. Anim. Behav. Sci. 2017; 187: 93–102. Publisher Full Text\n\nDeakin AG, Buckley J, Al Zubi HS, et al.: Automated monitoring of behaviour in zebrafish after invasive procedures. Sci. Rep. 2019; 21: 9.\n\nDe Lombaert M, Rick EL, Krugner-Higby LA, et al.: Behavioral characteristics of adult zebrafish (Danio rerio) after MS222 anesthesia for fin excision. J. Am. Assoc. Lab. Anim. Sci. 2017; 56: 377–381. PubMed Abstract\n\nBreacker C, Barber I, Norton WH, et al.: A low-cost method of skin swabbing for the collection of DNA samples from small laboratory fish. Zebrafish. 2017; 14: 35–41. PubMed Abstract | Publisher Full Text | Free Full Text\n\nTilley CA, Carreno GH, Sebire M, et al.: Skin swabbing is a refined technique to collect DNA from model fish species. Sci. Rep. 2020; 10: 18212. PubMed Abstract | Publisher Full Text | Free Full Text\n\nTilley CA, Barber I, Norton WHJ: Skin swabbing protocol to collect DNA sample from small-bodied fish species. F1000 Res. 2021; 10: 1064. Publisher Full Text\n\nBarber I, Arnott SA: Split-clutch IVF: A technique to examine indirect fitness consequences of mate preferences in sticklebacks. Behaviour. 2000; 137: 1129–1140. Publisher Full Text\n\nScott A, Sheldrick E, Flint A: Measurement of 17α, 20β-dihydroxy-4-pregnen-3-one in plasma of trout (Salmo gairdneri Richardson): seasonal changes and response to salmon pituitary extract. Gen. Comp. Endocrinol. 1982; 46: 444–451. PubMed Abstract | Publisher Full Text\n\nPottinger TG, Carrick TR: Stress responsiveness affects dominant-subordinate relationships in rainbow trout. Horm. Behav. 2001; 40(3): 419–427. PubMed Abstract | Publisher Full Text\n\nLivak KJ, Schmittgen TD: Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods. 2001; 25(4): 402–408. Publisher Full Text\n\nLenth RV: Emmeans: estimated marginal means, aka least-squares means.2021.Reference Source\n\nNorton W: Lidocaine and water conditioners.2022, May 10. Publisher Full Text\n\nTilley C: ARRIVE Author Checklist Validating skin swabbing as a refined technique to collect DNA from small-bodied fish species.pdf. figshare. Online resource.2022. Publisher Full Text"
}
|
[
{
"id": "159522",
"date": "02 Feb 2023",
"name": "Johan Ledin",
"expertise": [
"Reviewer Expertise Developmental biology"
],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe article ”Validating skin swabbing as a refined technique to collect DNA from small-bodied fish species\" is an article that evaluate techniques for tissue sampling of zebrafish and stickleback. Although fin clip surgery is relatively mild, the sheer number of interventions done world-wide for genotyping purposes makes this study important. This article compares fin clipping with skin swabbing – with and without the analgesic lidocaine. A major strength of the study is the combination of readouts, following each procedure, for behavior, cortisol and expression of stress genes which gives a rigid foundation for conclusions. The results suggest that skin swabbing is a true refinement compared to fin clip.\nWhat I would have liked, since the results are relevant for zebrafish researchers from all disciplines, is some further discussion on how big of a refinement this may be for the fish? Can the refinement be put in context with suffering of other procedures?\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate? Yes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes",
"responses": []
},
{
"id": "159524",
"date": "16 Feb 2023",
"name": "Marco Vindas",
"expertise": [
"Reviewer Expertise Behavioral neurobiology of fish (molecular biology",
"histology",
"behavior",
"stress and welfare)"
],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address 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 Tilley et al. is very good and builds on previous findings from the consortium that are replicated and corroborated in this work. In addition, the authors show some novel findings as well. In general, this manuscript is well written and clear. I have some minor comments and suggestions.\nResearch highlights\nUsing skin swabs instead of fin clipping is already a great benefit, but I don’t understand why it would be possible to use fewer animals? Wouldn’t the input for DNA collection be the same if one was using one or the other technique?\n\nHow speculative is the possibility of using this technique for the analysis of small metabolites?\nIntroduction\nThe authors refer to their findings towards the end of the introduction regarding lidocaine affecting fish for significant periods of time, but it is bit vague here. Could it be possible to just add a brief explanation of what they mean, for example that gene expression markers associated with their stress response are altered?\nMethods\nPlease include age and sex of the fish is missing for all groups of fish used in the experiments.\n\nGene expression analysis: which tissue was used in the analysis of gene expression trends?\n\nPrimer design: it appears as the authors designed some of the primers used in this study themselves. Did they also sequenced the finished product to make sure that they are obtaining results on the correct gene?\n\nStatistical analysis: why use two different statistical software? In general the R statistical program is more robust than others (such as prism) since one can include multiple parameters into the statistical model and therefore obtain a better explanation of the variation in the analyzed parameters. In addition, in terms of consistency, it would be better to only use one of them. Why were the FBI values not statistically analyzed?\n\nResults and discussion\nThe authors mention that fin clipped fish treated with lidocaine did not show a decrease in the FBI score similar to that shown by fin clipped fish with no lidocaine, which is great. However, they also write that the FBI score were not normalized after 6 h and therefore lidocaine might only be a short-term analgesic for this procedure. This is an important finding, but I struggle to see this in the data provided. There are no standard deviation bars or median values in figure 1B and no stats showing if there is a difference between pre and 6 h on the lidocaine fin clipped group and therefore hard to assess the aforementioned statement. Can the authors provide some more information here and better yet conduct a statistical analysis on the FBI data, which includes comparisons between treatment groups and timepoints?\n\nSince the authors present the behavioral FBI data first, it would make more sense to change figure 1 so that 1A shows the FBI data and 1B cortisol.\n\nThe gene expression data is very interesting, but it may be hard for the reader to understand what changes in these genes mean. Would it be possible to include a short explanation of what these genes are associated with and what it might mean that this is up or down regulated? For example, brain derived neurotropic factor is a marker for brain plasticity, it shows an increase to acute stress and a decrease to chronic stress. Overall low levels of bdnf are associated with bad welfare (i.e. decreased brain health).\n\nEven though its hard to compare the data across experiments, since they are conducted in different ways. Would it be possible for the authors to speculate on what the changes in gene expression means, when taking into consideration the cortisol and behavioral results from the first experiment?\nFigures\nIn order to make the data easier to understand. Can the authors illustrate the different treatments with different symbols in Fig 1B? It would also be good to include statistics within the figure so as to make it easier to assess differences between the groups (both treatments and time points).\n\nIt would be a bit more practical if the authors included a box within Figure 2 showing what C, SS and FC mean, this way one could just look at the figure without having to consult the text first. It would also be nice if the authors could include an illustration for the stats so as to show which groups are significantly different from each other (such as small letters), since this is not very clear if one just looks at the figures as they are.\n\nFor consistency, can the authors illustrate Fig 1A and the gene expression figs the same way?\n\nAre the 3Rs implications of the work described accurately? Yes\n\nAre a suitable application and appropriate end-users identified? Yes\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate? Partly\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes",
"responses": []
}
] | 1
|
https://f1000research.com/articles/12-28
|
https://f1000research.com/articles/12-27/v1
|
09 Jan 23
|
{
"type": "Research Article",
"title": "The future of Arabic language learning for non-Muslims as an actualization of Wasathiyah Islam in Indonesia",
"authors": [
"Mahyudin Ritonga",
"Sri Wahyuni",
"Hendri Novigator",
"Sri Wahyuni",
"Hendri Novigator"
],
"abstract": "Background: The majority of Indonesians believe and act as if the only people who can learn and be taught Arabic are Muslims. This fact goes against the basic idea that language is a way to communicate. So, the goal of this study is to find out what opportunities and plans there are for non-Muslims in Indonesia to learn Arabic. The goal of this study is to get language back to its basic function of being a way to communicate. It can also be the basis for putting Wasathiyah Islam into practise in Indonesia. Methods: This study was carried out in accordance with the official guidelines for research ethics established by the Ministry of Higher Education Research in the Republic of Indonesia. Between June 11 and August 30, 2022, the research was done. The research was carried out with two approaches, namely quantitative first and followed by qualitative. After collecting and analysing data for a study, researchers submit their findings to reviewers appointed by Indonesia's Ministry of Research and Technology. Results: A total of 64 participants were surveyed, and based on their responses, the following was determined; Researchers conduct interviews directly with informants who are considered to have the ability to provide information related to research themes. Therefore, researchers conducted interviews with 24. First, non-Muslims in Indonesia should study Arabic since language is a communication instrument. This includes religion, ethnicity, and race not limiting language acquisition. Second, teaching non-Muslims Arabic in Indonesia will help to implement Wasathiyah Islam there. Non-Indonesian Muslims who know Arabic will be more tolerant. Third, because Arabic is a communication tool, there is no big challenge for non-Muslims to learn Arabic. Conclusion: However, in terms of obstacles, of course, there are many big obstacles, namely the unsupportive Arabic learning.",
"keywords": [
"Arabic Language",
"Non-Muslim",
"Islam Wasathiyyah"
],
"content": "Introduction\n\nArabic is one of the compulsory subjects in Islamic Madrasah and universities in Indonesia which has been around for several years. For Indonesia, the role of Arabic is considered strategic in diplomatic affairs with Middle Eastern countries in the fields of education, politics, business, health, and the economy.1 This interest also refutes the narrative that states the limitations of Arabic only as a religious language in Islam. In fact, Arabic has the same function as other languages as a communication tool that can be used by everyone regardless of religion, ethnicity, culture, and race.2 However, in Indonesia, most of the objects of Arabic learning are only for Muslim students while non-Muslims are not involved.3 In point of fact, the goal of studying Arabic is to enhance one’s communication abilities in addition to gaining an understanding of the information included within the Qur’an, which is regarded by Muslims as their holy book.4 This is of course in line with the basic function of language as a communication tool so that Arabic can also be taught to non-Muslims in Indonesia.\n\nLearning Arabic for non-Muslims is part of the reflection on grounding Arabic as an international language.5 Arabic is now also the sixth official language of the United Nations since 1973.5,6 In addition, Arabic is also used as the official language of the Organization of African Unity (OPA). Arabic is now used as the official language of the Islamic World League (Rabithah Alam Islam), and the Organization of the Islamic Conference (OKI) which consists of 45 Islamic or Muslim-majority countries.6,7 However, that does not mean that Arabic is only used by Muslims but also by non-Muslims.8 It is known that the Urubah region, an area that includes 21 Arab countries including Arab Africa, Arab Asia, and the Arabian Gulf belonging to the Arab League with the official language of Arabic, does not entirely embrace Islam. However, Arabic cannot be separated from Islam because the main sources of law in Islam are the Al-Quran and Al-Hadith, both of which are in Arabic.7 This fact implies that Arabic can play a role in understanding the concept of Wasathiyah Islam in depth for Muslims and non-Muslims because access to learning Arabic is open to all religions.9\n\nA simple understanding of Wasathiyah Islam is the freedom to practice one’s beliefs according to one’s own religion, not leaning to the right or left. Islam Wasathiyah contains the meaning of progressive Islam.10 In the context of Islamic education and teaching, Wasathiyah means a consistent attitude that combines the Qur’an with the realities of life in accordance with the times.11 That is, to actualize Wasathiyah Islam in national and state life, one must mix the contents of the Qur’an with the context or reality of social life, making it immensely useful in constructing an Islamic image that is full of comfort and distant from all that is upsetting.12 Today, learning Arabic for all religions is considered appropriate to be a means of presenting a complete understanding of Wasathiyah Islam13 because many terminology in the Islamic concept of Wasathiyah are expressed in Arabic such as ummatanwashatan (Qur’an 2:143), middle way (tawasuth) and, being fair (’tidal), balanced (tawazun), tolerance (tasamuh), and many more. The actualization of Wasathiyah Islam will be much larger in Indonesia if every element of society has a comprehensive understanding and is committed to maintaining harmony between religious communities.14\n\nBased on the above considerations, learning Arabic for non-Muslims in the future becomes a topic that deserves serious discussion.5 So far, the classification of research on Arabic learning based on the characteristics of Wasathiyah Islam is still limited in the material aspect.15 Other studies also try to discuss the selection of the right media for campaigning Islam Wasathiyah in learning Arabic.16 The last is research on the challenges of learning Arabic based on Wasathiyah Islamic values.16 However, no fundamental research has been found on the future of learning Arabic for non-Muslims as the actualization of Wasathiyah Islam in Indonesia. As a sovereign country with the largest number of Muslims in the world, it is time for Indonesia to enable access to Arabic learning for non-Muslim students.17 This is a concrete effort from the government in implementing16 Wasathiyah Islam and as a support for Arabic as an international language that must be mastered by every Indonesian student to compete in the global arena.\n\nHowever, teaching Arabic for non-Muslim students with the purpose of actualizing Wasathiyah Islam is not easy because it will create a number of challenges and obstacles. This is natural because of the position of Arabic for non-Muslims in Indonesia as a second language which has many differences in language structure between Indonesian (their mother tongue) and Arabic (as a foreign language).8 Learning a language is the same as learning a culture.18,19 When a person learns Arabic, he or she is studying part of Arabic culture because the language is adopted from the culture and culture that developed in that area.18 Therefore, teaching Arabic to non-Muslims is like teaching them Arabic culture through the language aspect as a means of communication which can then be developed into a broad understanding of culture.18,20 When non-Muslim students can understand Arabic fluently, then they already have the important capital to understand the Islamic values so that they have a basis in actualizing Wasathiyah Islam properly.\n\nThe push for massive technological developments should make it easier for everyone to learn Arabic through online media that is connected to a computer or smartphone.19 However, the reality states otherwise as there are still Arabic students and teachers who cannot use technology in the learning process.21 In this condition, Arabic could be considered the object to blame because it is considered a difficult, boring, lagging, and monotonous lesson in comparison to other languages.20 In fact, there are no significant difficulties in learning Arabic as long as students and teachers are willing to dig deeper into information, innovate, and maximize the function of technology as a learning medium.22 Today, technology has also been used by some people to campaign for words using Arabic complete with translations to make it easier for everyone to understand the sentence,23 such as qul al-haq walau kana murran (say the truth even though it is bitter). This shows that Arabic is not anti-non-Muslim, but that on the contrary Arabic can be well received by non-Muslims.\n\n\nMethods\n\nEthical procedures were pertained by getting the approval from the ethical committee concerned to carry out the current study. Moreover, the inclusion of papers in this research was systematically done according to some keywords criteria mentioned below. All research works consulted were included herein and aptly cited. Finally, the findings were systematically presented; no bias by the researcher was allowed to interfere in an attempt to avoid affecting the findings of the study. As per procedure, the Scientific and Ethical Approval Committee in the Language Centre Institute, Muhammadiyah University of West Sumatra gave its consent for this research after its preliminary evaluation (Lembaga Penlitian dan Pengabdian Masyarakat (LPPM) /Nomor: 100/LPPM.UMSB/ST/09/2022-I). Following the mandated steps, the researcher submitted this to the Head of the English Department who facilitated the scheduling of a time and place for the researcher to acquaint the respondents with the study and its aims. Subsequently, written informed consent for publication of responses was also obtained by the researcher from the participants prior to participation, thus fulfilling the ethical requirements.\n\nThis research was conducted through stages that are in accordance with research ethics set by the Ministry of Higher Education Research of the Republic of Indonesia. Therefore, the steps taken are to start from the initial observation related to the focus in this study. After that, the team submitted a proposal to the ministry of higher education research to be considered as a recipient of funds, this stage took place from December 2021-May 2022. In May 2022, the Ministry of Higher Education then announced the nomination of the winner of the research grant that received funding, and the University then entered into an agreement with the Region X Higher Education Service Institution (LLDIKTI-X) to find an agreement related to the completion of research by the lecturer receiving the research fund. The University’s contract with LLDIKTI-X was then derived with a contract between the University through the Institute for Research and Community Service (LPPM) and the head of the research team.\n\nThis research uses a mix method or mixed methods. The design of this research was used sequential explanatory design, which combines quantitative and qualitative approaches sequentially. The first stage was carried out using a quantitative approach and the second stage was carried out using a qualitative approach.24 The quantitative approach used is in the form of a survey where the researcher conducted a survey to the respondents who are the research sample. Meanwhile, the qualitative approach used focused interviews which are described descriptively.\n\nParticipants are identified based on their ability to provide information related to the data needed according to the research theme, i.e., the teaching and learning of Arabic. Therefore, the heads of al-Islam study institutions, university leaders in charge of Al-Islam, lecturers who teach Al-Islam courses, and Arabic lecturers were identified as the right participants for this research theme. All participants were interviewed directly according to the content of the material and the data needed from them. This research was conducted through stages that are officially in accordance with research ethics set by the Ministry of Higher Education Research of the Republic of Indonesia. Therefore, the steps taken are to start from the initial observation related to the focus under study. After that, the team submitted a proposal to the ministry of higher education research to be considered as a recipient of funds this stage took place from December 2021 to May 2022. In May 2022, the ministry of higher education then announced the nomination of the winner of the research grant that received funding, and the University then enter into an agreement with the Region X Higher Education Service Institution (LLDIKTI X) to find an agreement related to the completion of research by the lecturer receiving the research fund. The University’s contract with LLDIKTI X was then derived with a contract between the University through the Institute for Research and Community Service (LPPM) and the head of the research team. In this case, the team through the head makes a statement of the ability.\n\nThe signing of the research contract between the Head of LPPM and the Head of the research team shows that teamwork can officially and legally begin. Therefore, the formal legality of this research began on June 10, 2022 in accordance with Contract No. 20/LPPM UMSB/SK P/06/2022. Since the signing of the contract between LPPM and the research team, researchers are given permission to collect data according to the established method. The study was conducted from 11 June 2022 to 30 August 2022. The researcher requested the willingness of the informants to provide the required data. The research data has been compiled and analysed is then presented before reviewers assigned by the ministry of higher education of the Republic of Indonesia. The results of the study are compiled in the form of a research report and written in the form of an article. To ensure the content contained, LPPM on Muhammadiyah University of West Sumatra establishes ethics in accordance with those applicable in the Ministry of Research and Higher Education of the Republic of Indonesia.\n\nResearchers conduct surveys as a technique for collecting data. The survey instrument is first arranged according to the research content. The instrument survey that has been prepared is then validated by 2 (two) experts, namely one expert in the field of Wasathiyah-based Islamic education and one more expert in the field of Arabic language education. The two experts provided notes related to the instrument content that had been compiled, the notes and recommendations of the two experts were used as a reference in improving the survey instrument. After the survey instrument is corrected, the researcher then disseminates it through the google form link that has been provided.\n\nBefore filling out the survey that has been distributed through the google form, participants must fill out a statement of willingness to respond to the statement that has been prepared objectively. After they fill in the statement, then they can continue to fill in and respond to the instruments that have been provided. Participants are given the opportunity to fill out the survey according to the predetermined time. The response from these participants is then automatically documented in the researcher’s email. A survey was used for the quantitative approach. Meanwhile, the qualitative approach used focused interviews which are described descriptively. Using Microsoft Excel, a descriptive analysis and processing is performed on all of the data that was obtained. The kind of this research is conducted to obtain more comprehensive data on ‘The Future of Arabic Language Learning for Non-Muslims as the Actualization of Wasathiyah Islam in Indonesia’ because it integrates the benefits of the two methods. The participants who filled out the survey with all the statements and answers via the email were 64 people. Meanwhile, the informants who provided data through interviews were 24 people, consisting of leaders of Al-Islam study institutions, university leaders in charge of Al-Islam, lecturers in the field of Al-Islam, and Arabic lecturers. The sampling technique of this research used was a cluster random sampling technique combined with convenience sampling, meaning that the sample is taken at random and also selected based on the availability of respondents and the ease of obtaining data. The data obtained through the survey as entered in the email is then randomized based on campus origin, study program, and religion. The formula used is RAND, the use of the formula is to ensure the percentage of those who fill out the survey based on campus origin, study program and religion, these three aspects are important to know because of the research content that aims to know the future of Arabic learning for non-Muslims.\n\nResearchers conduct surveys as a technique for collecting data. The survey instrument is first arranged according to the research content. The instrument survey that has been prepared is then validated by two experts, namely one expert in the field of Wasathiyah-based Islamic education and one in the field of Arabic language education. The two experts provided notes and recommendations related to the instrument content that had been compiled, these were used as a reference in improving the survey instrument. After the survey instrument was corrected, the researcher then disseminates it through the google form link that has been provided.25\n\nBefore filling out the survey that has been distributed through the google form, participants must fill out a statement of consent about the study and their participation. After they fill in the statement, then they can continue to respond to the survey that was then provided. The survey was circulated in Indonesian. Participants are given the opportunity to fill out the survey according to the predetermined time. Each question was presented as a ‘yes’ or ‘no’ response that participants could select. The response from these participants was then automatically documented in the researcher’s email. Quantitative data was obtained from distributing questionnaires via Google forms.25\n\nThe interview qualitative data was obtained through Google Forms and interviews. Researchers conducted interviews directly with 24 people. The questions were asked in person, and we shared the questions in Google forms. The data collected through interviews included the following:\n\n1. Arabic is the language of communication, not the language of religion\n\n2. Wasathiyah Islam and Arabic Learning Models for Non-Muslims\n\n3. Barriers and Challenges of Learning Arabic for Non-Muslims\n\nThe questions under the three research problems mentioned were in-person interviews with 24 participants, including seven Arabic language lecturers, eight Al-Islam lecturers, and nine Al-Islam university leaders. The researcher collected responses by writing down the answers. The researcher told them to provide their answers correctly and assured them that all of their information would remain anonymous and would not be used for other purposes.\n\nThe interview technique used was a structured interview, where the researcher used interview guidelines.25 The informants that were interviewed were not included in the informants surveyed, because the data required through the interview was different from the data collected through the survey. Interview informants were identified as those who are directly involved in setting curriculum policies in universities as well as those who carry out those policies. The data found based on the interview is set forth in the form of a record of the interview results.25\n\nThe survey took place from June 21, 2022 to August 31, 2022, within that time span participants are willing to be able to fill in each person’s instrument only once. The distribution of instruments is carried out through a google form, and the recap of filling out the survey is collected and documented directly to the researcher’s email. Completed surveys were sent to the researchers via email. The data extracted from the respondents were regarding: 1) Arabic as a communication tool, not a religious language; 2) Islam Wasathiyah and Arabic learning models for non-Muslims in Indonesia; 3) barriers and challenges of learning Arabic for non-Muslims in Indonesia.\n\nAnalysis of the quantitative survey data was performed by calculating a percentage of the responses, then the data were analysed using quantitative descriptive. Qualitative analysis consists of three steps after data collection: data condensation, data presentation, and conclusion drawing/verification.26,27 First, after the data was collected, the researcher classified the data based on the specified research problem. Second, the researcher presented the data according to the specified problem. Third, the researcher concluded the findings from the research problem. Based on the research findings, this analysis focused on the three problems that have been formulated and the data critically examined by following these three stages.28 The qualitative analysis methodologies of Miles and Huberman were used for this study.\n\nQualitative data were collected through the interviews and used to analyze and determine the research objectives. The data collected through interviews included Arabic as the language of communication, not the language of religion, Wasathiyah Islam and Arabic learning models for non-Muslims, barriers and challenges of learning Arabic for non-Muslims, students’ understanding of Islam instilled through learning Al-Islam and Muhammadiyah, and how non-Muslim students feel comfortable taking part in Al-Islam and Kemuhammadiyahan lectures. The interviews were conducted in four phases: Friday/July 15 2022, Saturday/23 July 2022, Monday/25 July 2022, and Wednesday/27 July 2022. The interview was conducted with 24 participants, including seven Arabic language lecturers, eight Al-Islam lecturers, and nine Al-Islam university leaders. The responses were collected by writing them down. It was assured that all their information would remain anonymous and would not be used for any other purpose by the researchers.\n\n1. Questions and responses related to (1) Arabic as the language of communication, not the language of religion, (2) interest in learning Arabic, (3) Arabic is a compulsory subject, (4) Arabic as the language of Muslim worship and the language of the holy book, (5) Arabic as an international language so Arabic is also important for people to understand, (6) a good understanding of Arabic makes individuals understand the contents of the Qur’an, and (7) that through technology people can more easily learn Arabic were related to the research problem that Arabic is the language of communication.\n\n2. The questions and responses related to (1) learning Arabic for non-Muslims as a part of the actualization of wasathiyyah Islam in Indonesia, (2) Indonesian non-Muslims also need to learn Arabic in order to better understand Islam wasathiyyah, (3) inclusion of Arabic in the general education curriculum in all schools/universities in Indonesia as part of wasathiyyah Islam, (4) living in harmony and peace between religious communities as the goal of the Islamic concept of wasathiyyah, (5) Arabic teaching materials between Muslims and non-Muslims must be distinguished were related to the Wasathiyah Islam and Arabic learning models for non-Muslims research problem.\n\n3. The questions and responses related to the (1) complexity of Arabic (such as aspects of sound, sentence structure, grammatical language) is an obstacle for individuals when learning Arabic, (2) motivation to learn Arabic, (3) lack of competence in Arabic is a big challenge for individuals to learn Arabic, (4) overcoming obstacles in learning Arabic, and (5) difficulty in learning Arabic with people of different religions are related to the barriers and challenges of learning Arabic for non-Muslim research problem.\n\n\nResults\n\nThe participants who filled out the research survey instrument were 64 people (Figures 1 and 2). If reviewed demographically, the classification is as follows:\n\nNote:\n\n1A: Arabic as a communication tool, not a religious language\n\n1B: Understanding Arabic well means you can also understand the Qur'an well\n\n1C: Arabic is an important international language to learn\n\n1D: Have a strong interest in learning Arabic\n\n1E: Arabic is a compulsory subject in schools/universities\n\nMeanwhile, the informants interviewed were a total of 24 people. The demographics of these informants are based on position on college and expertise. Therefore, the informant consists of five heads of Al-Islam study institutions in universities, five people come from leaders in charge of Al-Islam in universities. The experts interviewed were seven lecturers in the field of Al-Islam and seven lecturers in Arabic. Based on Figure 1, the sample of this study comes from several recognized religions in Indonesia. As many as 77% of respondents from Islam, 17% of respondents from Protestant Christianity and 6% of respondents from Catholicism, where respondents are classified from Islamic educational institutions such as madrasas, Islamic boarding schools, and Islamic universities and general education institutions such as public high schools to public college. The education level of the respondents is 5.3% from high school education level, 42.1% undergraduate level, 47.4% master level, and 5.3% doctoral level.\n\nLanguage is a tool used to express the contents of the heart, thoughts, ideas, and other ideas to the interlocutor. Through language a person can interact with each other in the living environment. This also applies to Arabic as an official communication tool used by many people to interact about their goals. However, many people also have heterogeneous viewpoints. According to them, Arabic is the language of a religion, because the texts of the holy Qur’an are in Arabic. Others also mentioned that by learning Arabic, they also learn some Islamic knowledge as most of the Islamic sciences use Arabic as the predominant language. According to Hakkoum and Raghay,29 such a view cannot be blamed unilaterally, considering that it is true that the Qur’an and that some books in the area of Islamic science use Arabic only. However, it is easy to access information through technology so that someone can learn Arabic for communication needs rather than religious or scientific purposes. For more details, the researcher will present the responses of the informants and respondents with heterogeneous religious classifications regarding the position of Arabic for religious people in Indonesia as follows:\n\nThe survey above describes the position of Arabic in Indonesia as a communication tool or purely as the language of Islam (Figures 3 and 4). The questionnaire revealed different responses from the respondents where in point 1A 75.4% of respondents agreed that Arabic was positioned as a communication tool and 24.6% of respondents agreed that Arabic became the language of Islam in Indonesia. From these results it can be concluded that most respondents consider Arabic as a means of communication in Indonesia and a small proportion believe Arabic as a religious language. However, when looking at the results of interviews with respondents who became the research sample for interviews, it was found that many respondents thought that Arabic as a communication tool provided an explanation of the position of Arabic in Indonesia as an important means of communication as well as being the language of Muslims considering Indonesia has a majority Muslim population. We believe that people who have a good understanding of the Arabic language can also explore the contents of the Qur’an well.30 This means that everyone, whether Muslim or non-Muslim, has the opportunity to understand the contents of the Qur’an comprehensively.17 However, it is very unfortunate if someone claims to be a Muslim but is unable to understand the meaning of the Qur’an because he does not understand Arabic grammar.\n\nNote:\n\n2A: Learning Arabic for non-Muslims is part of the actualization of Islam Wasathiyah in Indonesia\n\n2B: Non-Muslims also need to learn Arabic in order to better understand the purpose of Islam Wasathiyah\n\n2C: Arabic is included in the general education curriculum in all schools/universities in Indonesia as part of Islam Wasathiyah\n\n2D: Living in harmony and peace between religious communities is the goal of the concept of Islam Wasathiyah\n\n2E: My parents and family do not forbid me from learning Arabic\n\n2F: Arabic teaching materials between Muslims and non-Muslims must be distinguished to make it easier for non-Muslim students to learn Arabic\n\n2G: Technological developments make it easier for every believer to learn Arabic in Indonesia\n\nNote:\n\n3A: Grammatical factors, word structures, and sound aspects are challenges when learning Arabic\n\n3B: My weak Arabic competence is a serious obstacle for me to learn Arabic\n\n3C: Environment is a supporting factor to learn Arabic\n\n3D: Respondents' skills in overcoming obstacles in learning Arabic\n\n3E: Respondent's negative stigma towards Arabic\n\nAs the opinion of the respondents is as follows:\n\n“Arabic in Indonesia occupies an important position. Arabic should be used as a means of communication and occupy a higher position than other international languages in Indonesia. In fact, the government should require Muslim students in Indonesia to learn Arabic in order to explore and find out the contents of the Qur’an and As-Sunnah”.25\n\nThe opinions of the participants were supported by those of other respondents. The position of Arabic in Indonesia needs to be studied more deeply and academics are important to campaign for Arabic to be able to occupy a position as a communication tool. From the data in point 1C, it can be seen that most of the respondents think that Arabic has occupied the position of an important international language to learn. Even Shlowiy and Saad asserted that if you count all the varieties of Arabic today,8 there are around 313 million Arabic speakers worldwide, with Arabic as the fifth official language in the United Nations assembly, and the fifth most widely spoken language globally after Mandarin, Spanish, English, and Hindi.31 The rapid development of the role and function of Arabic in the international world cannot be separated from the development of the global economic system in the Middle East.32 Therefore, Arabic plays an important role in that situation where the people there actually communicate with Arabic. Also expressed by one respondent about the role of the Arabic language in the international world is as follows:\n\n“Arabic is a communication tool for Arabs that have spread all over the world. Arabic is very helpful for the development of knowledge in the fields of business, work, and education. In some international seminars and conferences, it is necessary to use Arabic.”.25\n\nHowever, if we look at the aspect of interest in learning Arabic, it is still less attractive to many students in Indonesia. This can be seen from the survey results at point 1D, only 34% of respondents have a strong interest in learning Arabic, and some 66% of respondents do not have the will to learn Arabic. The researcher studied the reasons for respondents who stated that they had a weak interest in learning Arabic so that it was revealed that the lack of interest of Indonesian students in learning Arabic was often motivated by the stigma of society that saw Arabic as difficult to learn. Our study revealed that Arabic in Indonesia is not a favourite subject because it is difficult and backward to learn, not cool, or that even some people are still embarrassed to use Arabic because of environmental limitations.20 Of the 66% of respondents who have an interest in learning Arabic, they consider Arabic to be a global communication tool and can help them understand Arabic-language Islamic treasures. From the results of interviews with non-Muslim respondents, they admitted that they are interested in Arabic because Arabic is a unique language, and some of them are curious to reveal the contents of classic books written in Arabic (Question 3).25 Even non-Muslim respondents believed that good Arabic language skills can reveal the contents of the Qur’an comprehensively (Question 2). This is in line with the opinion of Nasier and Nurdianto who said that the depth of the meaning of the Qur’an can only be revealed by people who have a deep understanding of Arabic both in terms of meaning, language rules, and word structure.7,31\n\nArabic language skills are the key to unlocking the secrets in every meaning of the word of the Qur’an.33–35 However, the lack of parental encouragement and no government regulation that requires all students to learn Arabic makes the lack of interest in learning Arabic intensely. These two factors are behind the limited reach of the Arabic language in Indonesia.36 In public schools or universities, Arabic is only a subject of local content or foreign language of specialization, while in Islamic schools and colleges Arabic is a compulsory subject for all students.37–39 This condition will slowly rule out Arabic compared to other international languages such as English which is a compulsory subject for all interfaith students. However, many respondents support Arabic as a compulsory subject from elementary school to university level, in its function as a communication tool (Question 1). A few respondents also refused to teach Arabic intensively for non-Muslim students, it was enough to include it in extracurricular activities so that those who were interested in Arabic could learn through these facilities. According to Carroll and friends Arabic in extracurricular activities has been around for a long time and interest in learning Arabic is still minimal, so the researcher suggests including Arabic in the list of compulsory subjects for all interfaith students based on the considerations above.40\n\nBasically, the actualization of Islam Wasathiyah in learning Arabic has existed since Islam came to Indonesia, it’s just that the Islamic Wasathiyah campaign has only emerged recently so it seems like a new issue.41 From the beginning of the spread of Islam in Indonesia around the 7th century AD, Islamic teachings were spread by Arabs living on the west coast of Sumatra in Barus or Fansur villages using Arabic script books.42,43 Sya’bani describe the presence of Islam carries a message of conditioning human hearts as a religion of rahmatanlil’alamin to this day as the purpose of strengthening Wasathiyah Islam is basically to create order in religious communities, protect the rights of religious adherents in exercising freedom of religion, create peace and peace in religious life and to realize the welfare of religious people.14\n\nAt point 2B not many of the respondents support that non-Muslims only need to learn Arabic as a form of understanding against Washatiyah Islam. In this case, the researcher looked at the responses from the respondents, to determine that those learning Arabic has no interest in anything related to religion. According to them, a person’s need in learning a language is only for daily communication and interaction when needed. Only 30% of the respondents think that non-Muslims need to learn Arabic to understand Wasathiyah Islam. This is also in accordance with the 2C aspect, namely regarding the Arabic language curriculum being included in general education. Many of the respondents supported this, especially Muslim 77% of total respondents from both the survey and interviews. However, not for non-Muslim respondents. According to Hassan and Abdullah,10 Arabic may be included in the general education curriculum with the aim of learning Arabic as a necessity that can be used in the future, not only as part of Wasathiyah Islam. So that there is no discrimination for non-Muslims to learn Arabic for needs other than communication and the needs that are used.\n\nLearning Arabic in Indonesia faces serious obstacles and challenges.44 The inhibiting factor is even more real when faced with non-native speakers, both Muslim and non-Muslim. Not only for non-Muslims, but Muslims who use Arabic in religious rituals still encounter many obstacles and challenges in learning Arabic because Arabic is not a communication language that has many language environments in Indonesia. Kim said that language is a culture and a habit, the more accustomed to using Arabic in interacting and making it a culture, the Arabic language skills will form by itself.36 The emptiness of the Arabic language environment is certainly a difficulty for students to actualize Arabic in real life. Similar provisions also apply to non-Muslims when learning Arabic will need the right space to communicate Arabic.26,27 In the formulation of the second problem, the researcher will describe the results obtained regarding the obstacles and challenges of non-Muslim students in learning Arabic.\n\nBased on the picture above, it can be revealed that a number of elements contained in Arabic such as aspects of sound, sentence structure, grammatical, and many more, turned out to be obstacles for 56% of respondents. Other elements such as aspects of media use, selection of methods, strategies, learning styles, and curriculum used in fact face serious challenges and problems. This variety of problems will certainly have an impact on non-Muslim students in learning Arabic. 70% of the respondents felt that learning Arabic is a formidable challenge for them due to this. This challenge is triggered by the development of globalization which requires everyone to be skilled in operating technology, so that two skills must be mastered at once, namely Arabic language skills and technology use skills.\n\n\nDiscussion\n\nToday, there are at least three challenges and obstacles that non-Muslim students will face when they want to learn Arabic. First, the linguistic aspect, as the linguistic difference between Indonesian and Arabic causes its own difficulties for Indonesian non-Muslims in learning Arabic, plus Arabic writing system which has certain rules and pronunciation of letter sounds that have no equivalent in Indonesian, there is also material on semantic aspects that discuss changes in meaning, differences in syntactic and morphological levels where in Arabic there is a change in word form that has a different level of complexity. different from Indonesian. In Indonesian it is known as basic words which then undergo a morphological process (affixes, repetitions, compound words), but the morphological process in Arabic relies on root words (tsulatsi, ruba’i, and khumasi) known as tashrif.\n\nSecond, the curriculum aspect, as in Indonesia there are two systems for presenting the Arabic curriculum, namely the integrated curriculum and separated curriculum. Both systems are a challenge for non-Muslim students because there are no regulations governing the Arabic language curriculum for non-Muslims either in madrasas or schools. In madrasas, in general they use an integrated curriculum, because Arabic is studied integrally as a separate subject that must be studied by every student. In schools, in general, Arabic is the subject of choice for students to improve foreign language skills. While in Islamic boarding schools, Arabic is usually studied through special subjects such as nahwu, sharaf, balaghah, fiqh, interpretation, and others. Third, the social aspect, the lack of sociological support for non-Muslims to learn Arabic is a challenge. This can be seen from the time Arabic was included in the national curriculum until now Arabic is still an exclusive subject for Muslims. In fact, researchers see the big role of Arabic being able to strengthen the massive actualization of Islam Wasathiyah in Indonesia if it is also taught to non-Muslims through this scientific study.\n\nIn addition to the above aspects, according to them, a supportive environment is also an important aspect in learning Arabic.45 However, as is known, the Arabic language environment in Indonesia is currently not widely found, especially for non-Muslims. In fact, in order to acquire the language more quickly and effectively, it is necessary to practice speaking Arabic directly. The Arabic environment referred to here is that there is no other language used in interacting except using Arabic. Because learning Arabic as a means of communication. If you want to be good at the language, it is recommended to live and interact with people who use Arabic as their daily language. The language environment greatly influences the process of one’s language development and can stimulate one’s interest and motivation in language. Madigan and friends assessed that 80% of a person’s language skills are shaped by environmental factors, while another 20% are obtained through theory.46\n\nHowever, another factor that they think is more important is the views and responses of the majority of people who consider Arabic as the identity of Islam in Indonesia. Of course, this kind of response is a challenge for non-Muslims to learn Arabic. It is different if Indonesia is not a Muslim majority so that the inhibiting factor for us is when we start learning Arabic. Most of the non-Muslim respondents in Indonesia are not familiar with Arabic even when speaking it. In addition, special Arabic language teachers for non-Muslims are not easy to find. Akmalia and friends stated that the vacancy or scarcity of Arabic language teachers for non-Muslims is due to the absence of regulations governing it such as Arabic subjects for non-Muslims, the scope of Arabic language materials for non-Muslims.47 Research data obtained by researchers found as many as 77% of respondents can overcome the obstacles they face. Abdulhameed explained that there will be many easy ways to learn Arabic if you have a strong will to learn, discipline, have a vision, and learn voluntarily without pressure and coercion.48 Moreover, technological sophistication can provide free Arabic learning services with selected materials according to the needs of learners that can be accessed anytime and anywhere.\n\nConsideration of the development of the Arabic language in Indonesia should also be used as a reason for teaching Arabic to non-Muslims. Moreover, Arabic in Indonesia has been studied since the time Islam entered Indonesia. Arabic has also been known by the Indonesian people since they knew Islam. However, Arifin et al., revealed until now the journey of Arabic in Indonesia is still limited to the interests of reading and understanding the Qur’an.3 Even if for the purpose of diplomacy with countries in the Middle East it can only be reached by a few people who have an interest as long as there is no policy from the government that makes Arabic a part of the national curriculum for all religious adherents. That way, along with the development of the times and the increasingly modern era of human needs, Arabic is not limited to one religion, but Arabic can also be used for the common good. Therefore, Arabic is not only learned by a group of Islamic religions in Indonesia, but also can be learned by all interfaith communities as a communication tool in interacting needs.\n\n\nConclusion\n\nThe results of this study indicate that: a) Arabic has been present in Indonesia since Islam entered Indonesia in the 7th century AD and from now until now Arabic is used as a communication tool to interact between one person and another about common goals and objectives. This is a strong argument that Arabic is not a religious language for Muslims in Indonesia even though Indonesia is the country with the largest number of Muslims in the world; b) the paradigm shift in Arabic as a means of communication in Indonesia makes Arabic relevant to be taught to non-Muslims, although this is a new proposal, it can be used as an instrument option for the actualization of Islam Wasathiyah in Indonesia. The government, in this case the Ministry of Religion, has an important role in making Arabic an inclusive lesson that is open to all interfaith students in the national curriculum. While the Arabic language learning model that can be used for non-Muslim students is through a multi-literacy learning model starting from basic literacy which discusses the basics of language skills (istima’, kalam, qira’ah, and kitabah), introduction of the sound system, ’alamattasykil, basic vocabulary, nahwu and shorof; c) Considering that language is something that must be learned, there are certainly obstacles and challenges for non-Muslims. Several obstacles were encountered such as pronunciation and writing that were not the same and also not commensurate with any language, changes in word form which also became changes in meaning, grammatical language which was quite complicated. Barriers like this are encountered because there is no intense learning carried out both at school and outside of school. This makes non-Muslims who learn Arabic feel challenged because there is no official regulation from the government that includes Arabic curriculum as a compulsory subject for non-Muslims. For non-Muslims who have never studied Arabic or even never know how the form of the Arabic part will be a big challenge for them in starting to learn Arabic.\n\nBased on the conclusions above from the survey results and also corroborated by interviews, this research is proof that Arabic is not only learned for Muslims. However, non-Muslims can also learn Arabic because according to him Arabic is the result of human culture that is used as a communication tool in interacting so that there are no limits for learners and their use. So that we as researchers suggest for further researchers to be able to develop this research, which is to present a suitable and appropriate learning model and its effectiveness has been tested for non-Muslims. So that in the future Arabic can become a preferred language lesson and become a need for the international community and can also be aligned with other international languages.",
"appendix": "Data availability\n\nFigshare: The future of Arabic language learning for non-Muslims as an actualization of wasathiyah Islam in Indonesia. https://doi.org/10.6084/m9.figshare.24066195.v1. 25\n\nThe project contains the following underlying data:\n\n• Complete underlying data.xlsx. (anonymised 64 survey responses).\n\n• Underlying interview data (anonymized).xlsx (anonymised 24 interviews responses).\n\nFigshare: The future of Arabic language learning for non-Muslims as an actualization of wasathiyah Islam in Indonesia. https://doi.org/10.6084/m9.figshare.24066195.v1. 25\n\nThis project contains the following extended data:\n\n• Arabic language survey questions. (Survey questions used in this study in English language)\n\n• Arabic language survey questions. (Survey questions used in this study in Indonesian language)\n\n• Interview Questoinnaire - English.docx. (Blank copy of the interview questions used in this study in English language)\n\n• Interview Questoinnaire - Indonesian.docx. (Blank copy of the interview questions used in this study in Indonesian language)\n\nRepository: COREQ checklist for ‘[The future of Arabic language learning for non-Muslims as an actualization of Wasathiyah Islam in Indonesia]’. https://doi.org/10.6084/m9.figshare.24066195.v1. 25\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\nPiazza BA: The Foreign Policy of Post-Mubarak Egypt and the Strengthening of Relations with Saudi Arabia: Balancing Between Economic Vulnerability and Regional and Regime Security. Handb. Socioling. 2017; 24(3): 401–425. Publisher Full Text\n\nFishman JA: Language and Ethnicity: The View from Within. Handb. Socioling. 2007; 453–460. 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Publisher Full Text\n\nCarroll KS, Al Kahwaji B, Litz D: Triglossia and Promoting Arabic Literacy in the United Arab Emirates. Lang. Cult. Curric. 2017; 30(3): 317–332. Publisher Full Text\n\nChaplin C: Salafi Islamic piety as civic activism: Wahdah Islamiyah and diffentiated citizenship in Indonesia. Citizsh. Stud. 2018; 22(2): 208–223. Publisher Full Text\n\nManshur FM, Husni H: Promoting Religious Moderation through Literary-based Learning. A Quasi-Experimental Study. Int. J. Adv. Sci. Technol. 2020; 29(6): 5849–5855.\n\nIryani E, Masruri M: Strategic Management of Change In Islamic Education Institutions (A Case Study at Islamic Education Institution of Syafana Islamic School Serpong). Dinasti International of Education Management And Soc. Sci. 2021; 295: 728–741.\n\nAbu-Absi S: The modernization of Arabic: Problems and prospects. Anthropological Linguistics. 1986; 28(3): 337–348.\n\nPikri F: The Role of the Language Environmment in Improving Arabic Learning Abilities. International Journal of Science and Society. 2022; 4(2): 346–354. Publisher Full Text\n\nMadigan S, McArthur BA, Anhorn C, et al.: Associations between Screen Use and Child Language Skills: A Systematic Review and Meta-analysis. JAMA Pediatr. 2020; 174(7): 665–675. PubMed Abstract | Publisher Full Text | Free Full Text\n\nAkmalia F, Zulfa YL, Kuraedah S: Teaching Arabic as A Heritage Language. Lang. Educ. 2022; 36(1): 95–98. Publisher Full Text\n\nAbdulhameed RS: Crimes Of Threats and Cyber Extortion Through social media: A Comparative Study. Rev. Int. Geogr. Educ. J. 2021; 11(12): 1022–1028. Publisher Full Text"
}
|
[
{
"id": "270692",
"date": "13 May 2024",
"name": "Aidillah Suja",
"expertise": [
"Reviewer Expertise Arabic Language Education",
"Teaching Arabic",
"Arabic Learning",
"Arabic Linguistic",
"Arabic as Foreign Language"
],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 an academic article titled \" The future of Arabic language learning for non-Muslims as an actualization of Wasathiyah Islam in Indonesia\" by Mahyudin Ritonga et al., the authors use a mixed methods approach combining quantitative surveys and qualitative interviews to explore the needs of Arabic language teaching for non-Muslims in Indonesia. Unfortunately, this article does not clearly present the research questions and objectives. This study followed the ethical research standards the Ministry of Higher Education Research of the Republic of Indonesia set. Using a sequential explanatory design, the researchers first collected quantitative data through a survey to assess general attitudes toward Arabic language learning for non-Muslims. This was followed by qualitative interviews that delved deeper into personal insights and experiences, thus providing a richer contextual understanding of the quantitative results. Although the authors provide considerable detail regarding methods and analysis, certain areas need more depth for full replication by other researchers. In particular, the descriptions of the collection, analysis of qualitative data, and selection of respondents could be improved by providing more detailed procedural information, including how the validity and reliability of the questionnaire and interview questions were tested. Improving these aspects would significantly increase the research's transparency and others' ability to replicate or develop this research in other contexts. It could be clearer in a study where quantitative and qualitative data are sourced from different subjects. Are there any references to this research model? The statistical analysis in this study was mostly descriptive, which is suitable for outlining basic trends and distributions in the data collected. However, this study did not use inferential statistics that could reveal deeper insights. The absence of non-Muslim informants in the qualitative data means that the data obtained needs to be more in-depth to explore information related to non-Muslims' needs for Arabic in the future. These limitations point to potential areas for further research that can use more robust analytical techniques to test specific hypotheses about Arabic language learning needs across different demographic groups. Data availability is another strength of this article, with the researchers stating that all data collected is accessible in the repository. This commitment to open data practices supports the reproducibility of the research. However, paragraphs are unnecessary repetition; the authors must pay attention to this to make the article more systematic. The data generally support the article's conclusions, although the broader applicability of these findings can only be questioned with further empirical evidence. Overall, this study provides a meaningful exploration of the potential for Arabic language learning to contribute to integration and social cohesion in Indonesia's pluralistic society. In conclusion, the findings of this study indicate that non-Muslims in Indonesia show interest in learning Arabic to enhance intercultural communication. These findings are interpreted within the framework of Wasathiyah Islam, which advocates the inclusion of Arabic in educational programs across religious boundaries to foster greater societal harmony and understanding.\n\nIs the work 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? Partly",
"responses": []
},
{
"id": "270693",
"date": "07 Jun 2024",
"name": "Yoke Suryadarma",
"expertise": [
"Reviewer Expertise Arabic Teaching and Learning",
"Teaching Arabic as second language",
"Arabic Linguistic",
"Arabic Corpus Linguistic."
],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nAccording to my thought, I have several review points. Firstly, this research has a specific objective. The objective of this research is to explore opportunities for non-Muslims to learn Arabic and promote communication through language, aligning with the principles of Wasathiyah Islam. In language education especially in Arabic, this research made a good contribution to Language Education in which this research sheds light on the importance of non-Muslims learning Arabic as a means of communication, potentially fostering greater societal harmony and understanding, once again, aligning with the principles of Wasathiyah Islam. But the research aims are not clearly mentioned in the introduction. In the introduction, it is not clear what exactly the researcher's purpose is in writing this study. It is as if this research is just to prove that Arabic is suitable and appropriate to be taught to non-Muslims for the reason of Wasahtiyatul Islam and the origin of the function of language is for communication. However, the formulation of the problem and the purpose of the research are not explicitly stated in the introduction. Secondly, this research has a good ethic research and an enough conducted method. The research has followed the official guidelines for research ethics established by the Ministry of Higher Education Research in the Republic of Indonesia, ensuring the study was conducted in an ethical manner. Not only that, this research also has utilized a mixed methods design, specifically a sequential explanatory design, combining quantitative and qualitative approaches to provide a comprehensive understanding of the topic. But There are inquiries to be made regarding the population and sample in this study. Based on the data obtained from interview questions in Data Availability, it appears that the population is exclusively comprised of a single Islamic university foundation, specifically the Muhammadiyah Universities. Furthermore, the specific campuses are not specified, despite the fact that Muhammadiyah Universities are highly abundant throughout Indonesia. How can this research be adapted to the Indonesian context? To ensure optimal and comprehensive results, it is necessary to conduct a demographic study throughout Indonesia at the Indonesian level. This study should include a representative sample from each island or province, encompassing various types of Islamic and non-Islamic colleges that offer Arabic language instruction. Subsequently, the sample size consisted of only 64 participants who completed the questionnaire and 24 participants who underwent interviews. Out of the total of 64 participants, 42 identified themselves as Muslims while the remaining 22 participants were non-Muslims. Therefore, the percentage of non-Muslims was only 34%. Given that this objective pertains to the future of Arabic language instruction, specifically its relevance to non-Muslims, it is recommended that the proportion of non-Muslim participants interviewed exceeds that of Muslims, ideally reaching at least 75%, rather than the opposite scenario. Third, this research really has comprehensive discussion. This research article has already presented a clear and comprehensive discussion. The discussion covers various aspects related to the importance of promoting Arabic language education for individuals of diverse religious backgrounds, the integration of Wasathiyah Islam principles, challenges and opportunities in language learning, educational policy implications, and the role of technology in language education. Fourthly, this research really has the accessible datasets. The datasets in this research are presented in a clear, and accessible format as written in the article like: Data Availability, Extended Data and Repository Link. The document mentions that the underlying data from the study, including survey responses and interview responses, are available on Figshare. Providing access to the underlying data on a reputable platform like Figshare enhances the transparency and accessibility of the datasets and it will be easy to be downloaded. In addition to the underlying data, the research article also includes extended data such as survey questions in English and Indonesian, interview questionnaires, and reporting guidelines. However, upon closer look, the interview questionnaires provided here are not simply a set of individual questionnaires. Rather, they resemble a brief research report, making them inappropriate for other researchers to reproduce. Fifthly, In the conclusion point, there are 3 conclusions conveyed by the researcher based on the results of the discussion, but actually the three conclusions are not included in the formulation of the problem raised by the researcher. This conclusion should contain the answer to the problem formulation presented by the researcher in the introduction. As addition, in this conclusion the researcher emphasizes that this research is proof that Arabic is not only learned by Muslims, but non-Muslims can also learn Arabic as a language of communication. This conclusion seems forced because in the discussion of the article there has not been seen any logical reason for non-Muslims to learn Arabic and what is also the reason beyond the reason for language as communication. Finally, Regarding the research results mentioned in the abstract, which suggest that Non-Indonesian Muslims who have an understanding of Arabic tend to be more tolerant, there is a lack of solid empirical evidence to support this claim.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Partly\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nNot applicable\n\nAre all the source data underlying the results available to ensure full reproducibility? Partly\n\nAre the conclusions drawn adequately supported by the results? Partly",
"responses": []
}
] | 1
|
https://f1000research.com/articles/12-27
|
https://f1000research.com/articles/11-583/v1
|
27 May 22
|
{
"type": "Research Article",
"title": "From head to rootlet: comparative transcriptomic analysis of a rhizocephalan barnacle Peltogaster reticulata (Crustacea: Rhizocephala)",
"authors": [
"Maksim Nesterenko",
"Aleksei Miroliubov",
"Aleksei Miroliubov"
],
"abstract": "Background: Rhizocephalan barnacles stand out in the diverse world of metazoan parasites. The body of a rhizocephalan female is modified beyond revealing any recognizable morphological features, consisting of the interna, the system of rootlets, and the externa, a sac-like reproductive body. Moreover, rhizocephalans have an outstanding ability to control their hosts, literally turning them into “zombies”. Despite all these amazing traits, there is no genomic and transcriptomic data about any Rhizocephala. Methods: We collected transcriptomes from four body parts of an adult female rhizocephalan Peltogaster reticulata: externa and main, growing, and thoracic parts of the interna. We used all prepared data for the de novo assembly of the reference transcriptome. Next, a set of encoded proteins was determined, the expression levels of protein-coding genes in different parts of the parasite body were calculated and lists of enriched bioprocesses were identified. We also in silico identified and analyzed sets of potential excretory / secretory proteins. Finally, we applied phylostratigraphy and evolutionary transcriptomics approaches to our data. Results: The assembled reference transcriptome included transcripts of 12,620 protein-coding genes and was the first for both P. reticulata and Rhizocephala. Based on the results obtained, the spatial heterogeneity of protein-coding genes expression in different regions of P. reticulata adult female body was established. The results of both transcriptomic analysis and histological studies indicated the presence of germ-like cells in the lumen of the interna. The potential molecular basis of the interaction between the nervous system of the host and the parasite's interna was also determined. Given the prolonged expression of development-associated genes, we suggest that rhizocephalans “got stuck in the metamorphosis”, even in their reproductive stage. Conclusions: The results of the first comparative transcriptomic analysis for Rhizocephala not only clarified but also expanded the existing ideas about the biology of this amazing parasites.",
"keywords": [
"Rhizocephala",
"parasitic barnacles",
"evolutionary transcriptomics",
"host manipulation",
"coloniality"
],
"content": "Introduction\n\nRhizocephalan barnacles (Crustacea: Rhizocephala) stand out among metazoan parasites. In the process of adaptation to a parasitic lifestyle, they have changed beyond recognition, losing almost all the structures characteristic of other crustaceans. In particular, they have lost all normal systems of organs, as well asbody axes1,2. The body of an adult rhizocephalan female is represented by the interna, a system of hollow, ramifying rootlets infiltrating the body cavity of the host (also crustaceans, usually decapods), and the externa, a sac-like body protruding outside the host. The interna is responsible for absorbing nutrients from the host hemolymph and their transportation to the externa1 as well as for interactions with the host3,4. The externa is a temporary structure thought to be an organ of sexual reproduction5. It usually contains two incorporated dwarf males and a special mantle chamber with developing embryos2. Some rhizocephalans can form several externae, sometimes as many as 2,0006,7, which is considered a unique instance of modular/colonial organization among arthropods. Besides their unusual morphology, rhizocephalans have evolved unique life cycle with a characteristic larval stage. The larva injects a few poorly differentiated cells into the host’s hemolymph, and what is left of the larva dies8. Noteworthy, the entire adult body originates from these cells and is thus a newly formed structure9.\n\nIn addition to their morphological adaptations and unusual life cycle, rhizocephalan barnacles show a remarkable ability for manipulating the host. These parasites can take control of the molting cycle of the hosts, change their metabolism, behaviour, and even body shape2,10–20. Specialized sites responsible for host-parasite interactions have recently been described3,4, with a network of the host’s neurons enlacing the rootlets of the parasite, but the molecular mechanisms of these interactions remained enigmatic. The authors suggested that the parasite may emit some signal molecules attracting the growth of the host’s neurons3.\n\nA rapid development of high-throughput sequencing technologies has enabled detailed molecular-based biological studies of many living organisms (for example, 21–25), but until recently, molecular studies on Rhizocephala have been lacking. The research on body heterogony, host-parasite interactions and functional physiology has only been based on morphological and other classical methods.\n\nIn an attempt to fill this gap, we made a comparative transcriptomics analysis of different parts of the rhizocephalan female body. Our research object was Peltogaster reticulata (Rhizocephala: Peltogasteridae), whose females parasitize hermit Pagurus minutus crabs (Crustacea: Decapoda) and form one or, less often, several externae. On the one hand, P. reticulata is a typical rhizocephalan, but, on the other hand, it has a lesser degree of modularity than many other representatives of this group26. Thus, it is a particularly convenient research model. In this study, we present the transcriptome-based evidence of molecular and functional heterogeneity of the female rhizocephalan body. We also show that the ovary is diffused throughout the interna, and host’s motor neuron axon are attracted to the interna rootlets. Phylostratigraphy and evolutionary transcriptomic analysis were performed for Rhizocephala for the first time. Our results make it possible to trace evolutionary trends in the P. reticulata gene set.\n\n\nMethods\n\nHermit crabs Pagurus minutus Hess, 1865 infected with Peltogaster reticulata Shiino, 1943 were collected in the Sea of Japan (Marine Biological Station “Vostok” of the Institute of Marine Biology of the Russian Academy of Sciences) (N: 42.893720, E: 132.732755). All the parasites were adults with fully developed externae.\n\nThe infected crabs were dissected in filtered sea water. The parasite was removed from the host’s body cavity and the remains of the host tissues were isolated from the interna. The body of each parasite was divided into four parts: 1) the externa, 2) distal part of the main trunk (the growing part), 3) the main part of the interna trunk (the main trunk), 4) the part of interna from the thorax of the host (the thoracic part) (Figure 1). For each body part the pooled sample was prepared, containing material from five parasitic individuals in two biological replicates. The samples were collected into the centrifuge tubes and frozen at -80 °C in IntactRNA (Evrogen, Moscow, Russia) reagent according to the manufacturer`s protocol.\n\n(a) Generalized scheme of female P. reticulata in the host. Colour sectors indicate the body parts examined in our study: externa (red), growing part of interna (green), main trunk of interna (purple), and thoracic part (blue). (b) The number of common OMA groups. The colour key on the heatmap shows the number of shared OMA groups between species. (c, e) Venn diagram for a set of genes either included in molecular signatures of the body parts (c) or over-expressed in the body parts (e). (d, f) Multidimensional scaling (MDS) plots for molecular signatures (d) and sets of over-expressed genes (f). Different clusters in MDS plots are marked with colours. Abbreviations: MS/overexp – the molecular signature or set of over-expressed genes for the body part, respectively; rep1/2 – biological replication identifier.\n\nBefore RNA isolation, the IntactRNA-fixed samples were rinsed in 0.1M phosphate-buffered saline (PBS). The total RNA was isolated using Quick-RNA MiniPrep (R1054, Zymo Research, Irvine, California, USA) according to the manufacturer protocol. The libraries were synthesized using NEBNext Ultra Directional RNA Library Prep Kit for Illumina (E7760, New England BioLabs, Ipswich, Massachusetts, USA) according to the manufacturer protocol. Paired-end sequencing was carried out using Illumina HiSeq 2500 instrument (Illumina, San-Diego, California, USA).\n\nSampling was conducted in accordance with the European Community Council Directive of November 24, 1986 (86/609/EEC). All possible effort was made to minimize the number of animals used.\n\nThe primary quality control of paired-end reads libraries was manually assessed using FastQC (v0.11.5) [https://www.bioinformatics.babraham.ac.uk/projects/fastqc/]. The potential sequencing error identification and correction was performed by Karect27 (v1.0) [https://github.com/aminallam/karect] with the following parameters: --celltype=diploid –matchtype=hamming. Trimmomatic (v0.39) [http://www.usadellab.org/cms/?page=trimmomatic] was used for removing the sequencing adaptors (ILLUMINACLIP:$ADAPTERS:2:30:10:2:TRUE), low-quality read regions (SLIDINGWINDOW:4:20 MAXINFO:50:0.8) as well as reads with a length less than 25 nucleotides (MINLEN:25). Since libraries preparation and sequencing were performed in the laboratory where researchers also work with human medical samples, we checked the parasite data for the presence of the read pairs with a high identity to the Homo sapiens reference transcriptome (GENCODE v.31) using BBTools (v37.02).\n\nThe prepared libraries were pooled and used for de novo reference transcriptome assembly using Trinity28 (v2.5.1) with k-mer size and required minimal contig length equal to 25 and 200 nucleotides, respectively. The assembled contigs were renamed by adding the four-digit tag of the species, “Pret”, at the beginning of IDs. Isoforms were clustered on all assembled contigs using CDHIT-est29 (v4.7) [http://weizhong-lab.ucsd.edu/cd-hit/] and a sequence identity threshold equal to 95% (-c 0.95), accurate mode (-g 1), and both +/+ and +/- strands alignments (-r 1). TransRate30 (v1.0.1) was used for the quality assessment of clustered sequences. Only the contigs classified as “good” by TransRate30 were included in further analysis.\n\nThe 18S and 28S ribosomal RNA sequences were searched using RNAmmer31 (v1.2) from the Trinotate pipeline (v3.1.1). The sequences obtained this way were compared with the NCBI nucleotide database with BLASTn32 (v2.6.0+) to identify their possible sources.\n\nWe used Model-based Categorical Sequence Clustering (MCSC) Decontamination method33 for removing potential contamination, with “Arthropoda” as the target taxon and the clustering level equal to 5 (32 clusters). The parsing of BAM-files with reads alignment results created by Bowtie234 was performed to extract only reads pairs that mapped to the decontaminated set of contigs.\n\nSalmon35 (v1.0.1) was used for expression level quantification (-l ISF –discardOrphans –seqBias –gcBias –validateMappings). The expression quantification results and the transcripts-to-genes map from the Trinity output were provided to the “tximport” package for R to obtain expression levels of genes. The tables with both unaveraged transcripts-per-million (TPM) values and TPM values averaged between biological replicates were prepared. Only the sequences with expression levels ≥ 1 TPM in at least one sample were included in further analysis.\n\nTransDecoder (v5.5.0) was used for the identification of the amino acid sequences encoded by assembled contigs. Firstly, the long open reading frames with a length ≥ 100 amino acids (aa) and products of its translation were found. Secondly, identified proteins were compared with the NCBI non-redundant (DIAMOND BLASTp36 (v0.9.22.123), e-value = 1e-3) and the PfamA37 (HMMscan (v3.1b2)) databases. Thirdly, the comparison results were provided to the TransDecoder to identify the likely coding regions and to obtain the probable set of proteins.\n\nIn our analysis, we focused only on genes that successfully passed two filters: 1) noticeable expression level (i.e., the expression is ≥ 1 TPM in at least one sample) and 2) encoding of the proteins with a length greater than or equal to 100 amino acids. Only the longest protein and its coding transcript were selected as representatives for each gene and referred to as “reference sets”. The completeness of the protein reference set was evaluated by comparison with the database of single-copy orthologues of Metazoa (odb-9) using BUSCO38,39 (v3.0.1) (e-values = 1e-3, mode = proteins).\n\nFor the annotation of the genes, their nucleotide and amino acid sequences were compared with publicly available databases: NCBI nucleotide collection (nt), NCBI non-redundant (nr), and SwissProt40. The similarity search was carried out with BLASTn megablast32 (nt) and the sensitive mode of DIAMOND BLASTp36 (amino acid databases), with an expected value (e-value) threshold equal to 1e-3 and a limit up to 10000 for the number of description and alignments41. The best BLAST hits (BBH) were selected with a custom script.\n\nThe potential domain architecture of the proteins was identified using the PfamA database (HMMScan) and custom script. The proteins were also analysed using the eggNOG-mapper web-resource42 (v2) [http://eggnog-mapper.embl.de] with default parameters.\n\nOrthogroups were identified with the use of OMA standalone program (v2.5.0)43 in three steps. Firstly, the reference proteomes of Amphibalanus amphitrite Darwin, 1854 (UP000440578), Armadillidium nasatum Budde-Lund, 1885 (UP000326759), Armadillidium vulgare Latreille, 1804 (UP000288706), Daphnia magna Straus, 1820 (Strain: Xinb3) (UP000076858), Daphnia pulex Leydig, 1860 (UP000000305), Penaeus vannamei Boone, 1931 (UP000283509), Portunus trituberculatus Miers, 1876 (UP000324222), and Tigriopus californicus Baker, 1912 (UP000318571) were downloaded from the UniProt40 database [accessed 21 October 2021]. Only the sequences with a length equal to or more than 100 amino acids were analysed. The OMA standalone was run with default parameters with the “bottom-up” algorithm for inference of hierarchical orthologous groups (HOGs), without a phylogenetic tree, but with the identification of two Daphnia species as an out-group. Secondly, we reconstructed the phylogenetic tree following the protocol by Dylus et al.44. Briefly, using the filter_groups.py provided, we selected OMA groups that included at least eight of the nine crustacean species involved in the analysis. Then, using MAFFT45 (v7.487), multiple protein alignment in each orthogroup was performed (--maxiterate 1000 -localpair). The alignments were concatenated into a supermatrix using the concat_alignments.py. The selection of suitable sites in the supermatrix was carried out using tramAl46 (-automated1). We used the ProtTest program47,48 (v3.4.2) to determine the most appropriate sequence evolution model. The phylogenetic tree was reconstructed using the IQ-TREE49,50 (v2.1.4-beta) with the following parameters: -m LG+I+G+F --seed 12345 -B 1000 --nmax 1000. The consensus tree was rooted by the out-group using the “ape”51 package for R. Thirdly, the phylogenetic tree was used when the OMA standalone43 was re-run with default settings. The construction of a heatmap with the number of common OMA groups between the studied species was performed in RStudio using the “ggplot2” (v3.3.5), “pheatmap” (v1.0.12), and “RColorBrewer” (v1.1-2) libraries.\n\nWe defined the “molecular signature” of a body part as a set of genes with an expression level ≥ 2 TPM in the body part. The expression threshold value was chosen in accordance with the results of studies by Wagner, Kin, and Lynch, according to which “genes with more than two transcripts per million transcripts (TPM) are highly likely from actively transcribed genes”52. If a gene had an expression ≥ 2 TPM in all the body parts, we classified it as “commonly expressed”.\n\nSignificant variation of gene expression between samples was detected using the “RNentropy” library53 (v1.2.2) for R. The analysis was carried out using a table with unaveraged TPM values between replicates. We used the corrected global sample specificity test P < 0.01 according to the Benjamini-Hochberg method, and local sample specificity test P < 0.01.\n\nThe overlaps between the molecular signatures and the sets of over-expressed genes were visualized with the InteractiVenn54.\n\nWe performed a multidimensional scaling (MDS) analysis for the molecular signatures and the sets of over-expressed genes. The presence / absence matrices were used as input. It was indicated in the matrix for each gene (row) whether the gene was included in the molecular signature of the body part or had an increased expression in it (“1”) or not (“0”). The optimal number of clusters was determined using the “silhouette” method implemented in the “factoextra” library (v1.0.7) for R. We used the metaMDS function from the “vegan” library (v2.5-7) with the following parameters: distance = \"manhattan\", try = 100, trymax = 100000, autotransform = FALSE, binary = TRUE, k = the optimal number of clusters. The seed was set to 1234 both when the optimal number of clusters was determined and in MDS. To visualize the results, we used the ggscatter function from “ggpubr” (v0.4.0) library for R.\n\nThe in silico identification of potential ESP was performed according to the pipelines described by Garg and Ranganathan55. Firstly, all proteins from the reference set were analysed with SignalP56 (v5.0b). Based on the analysis results, the proteins were divided into potential “classical” (SP ≥ 0.5) and “non-classical” (SP < 0.5) ESP. Secondly, the “non-classical” ESP were analysed using SecretomeP57 (v1.0) [https://services.healthtech.dtu.dk/service.php?SecretomeP-2.0]. Only proteins that had NN-scores ≥ 0.9 and were simultaneously predicted not to contain a signal peptide were selected. Thirdly, all potential ESP were scanned for the presence of the mitochondrial transit peptide with TargetP58 (v2.0). The proteins with this signal were excluded. Fourthly, transmembrane hidden Markov model (TMHMM)59 (v2.0c) [https://services.healthtech.dtu.dk/service.php?TMHMM-2.0] was used to model and predict the location and orientation of transmembrane domains in proteins, and only proteins without them were considered as potential ESP. Out of these potential ESP, we selected only those that were included in at least one molecular signature. The overlap analysis between ESP sets were performed using InteractiVenn54.\n\nWe ran all against all BLASTp searching using DIAMOND36 (--evalue 1e-3) for both classical and non-classical ESP. Similarity search results were used in Silix60 (v1.2.11) (-r 0.9), which assigns proteins to putative gene families. For annotation, all ESP were compared against the NeuroPep61 and MetazSecKB62 databases using DIAMOND BLASTp36 with the following parameters: --sensitive --max-target-seqs 10000 –evalue 1e-3. The best BLAST hits were selected using a custom script.\n\nThe GSEA using “topGO” library (v2.40.0) for R was performed for 1) whole molecular signatures, 2) sets of over-expressed genes, and 3) sets of potential “classical” and “non-classical” ESP. Only the Gene Ontology (GO) terms describing biological processes were considered. We used Fisher’s exact test and extracted only the terms including at least 10 significant genes (GO terms with p-value <0.01) from the results. Redundancy was reduced with the “rrvgo” library (v1.0.2) . We used the minus log10-transformed p-values as scores, org.Dm.eg.db as database, relevance as similarity measures methods, and 0.7 as the threshold for reduceSimMatrix. We used the “wordcloud” (v2.6) library for R to build word clouds based on the redundancy reduction results. The more often the parental bioprocess was found in the list of enriched bioprocesses, the larger the word size. Each bioprocess was assigned a colour from the “viridis” (v0.6.1) palette package.\n\nThe phylostratigraphy analysis of the P. reticulata reference protein set was performed using the “phylostratr” package63 (v0.2.1) for R. We used reference proteomes of Crustacea prepared beforehand as well as the prebuilt dataset of prokaryotes, human, and yeast. Similarity search between proteins was carried out with BLASTp32 (v2.6.0+). The tables with BLAST results in “6” output format were used as input for “phylostratr” for protein distribution between phylostrata: 1) “Cellular organisms”, 2) “Eukaryota”, 3) “Opisthokonta”, 4) “Metazoa”, 5) “Eumetazoa”, 6) “Bilateria”, 7) “Protostomia”, 8) “Ecdysozoa”, 9) “Panarthropoda”, 10) “Arthropoda”, 11) “Mandibulata”, 12) “Pancrustacea”, 13) “Crustacea”, 14) “Multicrustacea”, 15) “Hexanauplia”, 16) “Cirripedia”, and 17) “Peltogaster reticulata”.\n\nThe phylostratigraphic composition was analysed for P. reticulata reference gene set, the set of genes with noticeable (≥ 2 TPM) expression in all the female body parts considered, the sets of overexpressed genes as well as the sets of genes encoding potential “classical” and “non-classical” ESP. The results were visualized using “ggplot2”, “viridis” (v0.6.1), and “reshape” (v0.8.8) libraries for R.\n\nTranscriptome Age Index (TAI) definition was performed for P. reticulata body parts using phylostratigraphy results and tables with averaged TPM-values. The analysis was carried out using the “myTAI”64 (v0.9.3) package for R. Genes with an expression level < 2 TPM at all the body parts were excluded. The analysis was carried out on log2(TPM + 1) transformed values. We used the FlatLineTest function to quantify the statistical significance of the global TAI pattern. For analysis with the use PlotRE and PlotBarRE functions, the phylostrata were divided into two groups: “before” (phylostrata 1-13), and “after” (phylostrata 14-17) the division of Crustacea.\n\nUsing the pMatrix function from “myTAI”64, the contributions of genes to the TAI of body parts were determined. For each body part, 500 genes with the largest contribution were selected out of the genes with the GO annotation. Further, GSEA for the selected gene sets was performed similarly to GSEA for the molecular signature.\n\nFor histological and light-microscopic examination, the dissected internae were fixed with Bouin solution (picric acid (trinitrophenol) 71.5%, paraformaldehyde 24% and acetic acid 4.5%). Paraffin sections 5 μm thick were made using standard histological methods with the help of a Leica RM-2265 microtome and stained with hematoxylin-eosin. The sections were examined under a Leica DM2500 microscope. The photos were taken with a Nikon DS-Fi1 camera and processed with ImageJ software (FiJi65).\n\nSamples of interna for immune labelling were fixed with 4% paraformaldehyde (PFA; Sigma-Aldrich) in PBS (Fluka) at 4 °C for four hours, and then rinsed three times with PBS. Before the immunocytochemical staining, the fixed material was incubated with PBST (PBS + 0.1 % Triton-X100; Sigma-Aldrich) during 24 hours at 4 °C. Then, the samples were incubated in primary antibodies and anti-acetylated α-tubulin (Sigma Aldrich, Germany, T6793, produced in mice) and anti-serotonin (Sigma Aldrich, Germany, S5545, produced in rabbit) for three days. After incubation the specimens were rinsed in PBS three times and incubated in secondary antibodies anti-mouse IgG CFTM 633 (Sigma Aldrich, Germany, SAB4600138) and anti-rabbit IgG CFTM 488A (Sigma Aldrich, Germany, SAB4600030).\n\nThe specimens were rinsed with PBS three times and stained with the DAPI nuclei stain (1 μg/ml; Sigma-Aldrich) for 30 min, rinsed in PBS and mounted in DABCO-glycerol. The samples were examined using the confocal laser scanning microscope Leica TCS SP5 in the Resource Center “Microscopy and Microanalysis” of the Research Park of Saint Petersburg State University. The images were processed by ImageJ software (FiJi65).\n\nSpecimens for SEM were fixed at 4 °C in 2.5% glutaraldehyde, dehydrated in ethanol series and acetone, critical point-dried in a Hitachi critical point dryer HCP- 2, mounted on stubs, coated with platinum with the use of a Giko IB-5 Ion coater, and viewed under a FEI Quanta 250 scanning electron microscope in the “Taxon” Research Resource Center of the Zoological Institute of the Russian Academy of Sciences.\n\n\nResults\n\nThe transcriptomes of the whole body (Figure 1a), the thoracic part of the interna (Figure 1a), the growing part of the interna (Figure 1a) and the main trunk of the interna (Figure 1a) as well as that of the externa (Figure 1a) of adult P. reticulata were collected and sequenced in two biological replicates. More than 87% of read pairs remained in all the paired-end libraries after the removal of adapters, poor-quality regions, and short sequences (Table S1, Underlying data66). The potential contamination with human-derived sequences did not exceed 4.1% from the total number of trimmed read pairs in each library (Table S1, Underlying data).\n\nAll prepared libraries were merged, and the resulted libraries were used as input for Trinity software. In general, 353,130 contigs with lengths greater or equal to 200 nucleotides were assembled de novo. After the clusterization of similar sequences, the P. reticulata transcriptome included 267,188 contigs. TransRate assembly and optimal scores made up 0.3331 and 0.3835, respectively. More than 95% (256047/267188) of the contigs were well-assembled (“good”) according to the TransRate quality control results. The completeness analysis for “good” contigs using a database of the metazoan single-copy orthologues revealed that 96.3% (Single: 57.8%, duplicated: 38.5%) of the orthologues were assembled completely.\n\nGiven the parasitic lifestyle of P. reticulata, the assembled transcriptome was checked for the presence of potential contamination. According to the RNAmmer analysis results, eight and nine sequences could be classified as 18S and 28S ribosomal RNAs, respectively. The comparison with the NCBI nucleotide database revealed that the contigs successfully aligned with ribosomal sequences from Alveolata (HQ891115.2), Fungi (AY382649.1; CP033152.1; CP030254.1; GQ336996.1; MF611880.1), and Metazoa (AY265359.1; EU082415.1; KY454201.1; EU370441.1; KU052603.1). Among the metazoan hits, only sequences from Crustacea were identified. The identity percentage with the database-derived 18S host (KY454201.1) and parasite (EU082415.1) sequences made up 88% (990/1,123) and 99% (1,200 / 1,201), respectively. In this study, we used the MCSC hierarchical clustering algorithm for removing potential contamination from the assembled transcriptome. The decontaminated transcriptome included 80,779 contigs and contained 81.6% (Single: 56.9%; duplicated: 24.7%) of completely assembled single-copy metazoan orthologues. The number of paired-end reads successfully aligned to selected contigs varied from 10.74 (externa, first replicate) to 30.64 million (the thoracic part of the interna, second replicate) (Table S1, Underlying data)\n\nThe sequence expression level quantification was performed with Salmon by mapping selected read pairs to the decontaminated transcriptome. The mapping rate ranged from 85% to 91%. The transcript-to-gene map was used to obtain gene expression levels in TPM values. After excluding genes with a low activity in all analysed samples (expression level < 1 TPM), the dataset contained TPM values for 20,980 contigs. According to the TransDecoder results, 32,990 contigs encoded proteins with lengths ≥ 100 amino acids.\n\nOnly the protein-coding genes with a noticeable expression were involved in further analysis. After filtering by expression level (≥ 1 TPM in at least one sample) and encoded protein lengths (≥ 100 aa), we obtained the reference gene set for P. reticulata containing 12,620 sequences. For each gene, the longest protein encoded by its splice variants was selected as a representative sequence. The comparison with a single-copy metazoan orthologues database revealed that in the reference protein set of P. reticulata 75.6% of the orthologues were assembled completely, whereas 21% of the sequences were absent.\n\nThe prepared reference sets were compared with publicly available databases. According to the results, 3,399, 8,502, and 9,690 genes had hits with NCBI nucleotide, SwissProt, and NCBI non-redundant databases, respectively. The overlap analysis revealed that 3,240 genes were successfully annotated using each database. Moreover, 6,090 genes belonged to at least one GO terms. The domain architecture of the encoded protein was identified for 8,803 genes based on the comparison with the PfamA database. Annotation results are presented in Table S2 (Underlying data67).\n\nThe identification of orthogroups in P. reticulata and other Crustacea involved in this study was carried out in three stages. First, the proteomes of P. reticulata and eight reference crustacean species were analysed using OMA standalone. As a result, 24,840 OMA groups were discovered.\n\nSecondly, a phylogenetic tree of the studied crustacean species was constructed based on the results (Figure S1, Extended data68). We selected 609 OMA groups, containing at least eight out of nine species. The multiple protein alignment results in each of the OMA groups were concatenated into a supermatrix. After site selection, the final supermatrix contained 282,907 sites. In the resulting phylogenetic tree, P. reticulata was united into the same taxon with another cirripede barnacle, Amphibalanus amphitrite, with full support (Figure S1, Extended data68).\n\nThirdly, the resulting tree was used to refine the search results for orthogroups. More than 20,000 orthogroups were found: 24,840 and 20874 OMA and HOGs were identified, respectively. Figure 1b shows the number of common OMA groups for pairs of species. P. reticulata had the largest number of \"common\" OMA Groups (4,354) with A. amphitrite. For comparison, P. reticulata had no more than 3,490 “common” OMA groups with other crustacean species, the smallest overlap being found with Portunus trituberculatus (1,262 OMA groups).\n\nWe defined the molecular signature of a body part as a set of genes with an expression ≥ 2 TPM in the body part considered. Each molecular signature included at least 8,000 genes: the main trunk of interna (8,070 genes), the growing part (8,148), the thoracic part (8,223), and externa (9,233) (Table S3, Underlying data). Approximately 54% (6,829 / 12,620) of the genes from the reference set were included into molecular signatures of all body parts (Figure 1c). Figure 1c shows significant overlaps between the parts of the interna and an almost 10-fold difference in the number of “specific” genes between the interna parts and the externa. Based on the gene expression, the body parts were divided into two clusters (Figure 1d). The first cluster contained all parts of the interna, while the second cluster contained only two replicates of the externa.\n\nAccording to the differential expression analysis results, the number of over-expressed genes varied from 204 (the main trunk of the interna) to 2,224 (the externa) (Table S3, Underlying data). The number of genes with an increased expression in the interna did not exceed 283. Figure 1e shows that (i) only 1 gene was over-expressed both in the externa and in the interna, (ii) the number of “shared” over-expressed genes between different interna parts ≤ 35, (iii) only six genes were over-expressed in the whole interna. The externa clustered separately from the interna, and all interna parts were remote from each other (Figure 1f).\n\nThe results of identification of molecular signatures and differentially expressed genes are presented in Table S3 (Underlying data69).\n\nFigure 2a and b show Venn diagrams for lists of bioprocesses enriched with genes included in the molecular signatures (Figure 2a) and genes with increased expression (Figure 2b) in the female body parts considered. In contrast to the externa, where many active bioprocesses were associated with development, bioprocesses enriched in the interna were mainly connected with metabolism. Comparative analysis results revealed that 50 bioprocesses were active in at least two parts of the female body. Among them were “mitotic cell cycle process” (main trunk and the thoracic part of interna), “cell division” (growing and thoracic part of interna), “homeostasis of number of cells” (same), “apoptotic signalling pathway” (same), “symbiotic process” (growing and main trunk of interna), “determination of adult lifespan” (same), “immune system process” (the growing part, the main trunk, and thoracic part of interna), and “autophagy” (same).\n\n(a, b) Venn diagram for sets of parental bioprocesses enriched with either genes included in the molecular signature of the body part (a) or genes over-expressed in body part considered (b). (c) Clouds of enriched parental bioprocesses for the body parts. Most often, the bioprocess was found in the list of enriched bioprocesses, the larger the word size. Abbreviations: MS/Over – bioprocesses enriched with genes included either in molecular signature or in sets of over-expressed genes; PB – parental bioprocesses. (d–f) Histological sections of the main trunk of P. reticulata 1 - the wall of the main trunk; 2 - central lumen; 3 - groups of the floating cells; 4 - Nuage body. Scale bars: d - 200µm, e - 20µm, f - 50µm.\n\nThe “germ cell development” bioprocess was enriched in the main trunk and the growing part of the interna, whereas “female gamete generation” was only found among the lists of enriched bioprocesses in the thoracic interna part (Figure 2c). At the same time, according to our results, meiosis probably only occurred in the externa (Figure 2c). The results of histological studies also confirmed the presence of germ-like cells in the central lumen of the rootlets of the interna. Groups of floating small round cells with a high nuclear cytoplasmic ratio were found in the central lumen of the main trunk and peripheral rootlets (Figure 2d–f). A Nuage body (Figure 2d–f), which is a marker of germ cells, was present in each cell next to the nucleus.\n\nNo common bioprocess was found in different parts of the female body, enriched with over-expressed genes. The genes with over-expression in the externa were involved in bioprocesses associated with cuticle transformation and development of the nervous system. In the growing interna part, over-expressed genes were involved in “gland development,” “response to nutrient levels”, “organic acid transport”, as well as lipid and fatty acid metabolic processes. In addition to various metabolic processes, “determination of adult lifespan” and “intrinsic apoptotic signaling pathway” were also found among a set of enriched bioprocesses for the main trunk. Bioprocesses associated with responses to various stimulus (bacterium/oxygen-containing compound/wounding), as well as “interspecies interaction between organisms”, “ion transmembrane transport”, “cellular homeostasis”, and “aging” were classified as “enriched” in the thoracic part of the interna.\n\nAll GSEA results are presented in Table S4 (Extended data70).\n\nThe identification of potential ESP was performed in silico. We found 852 \"classical\" and 282 \"non-classical\" ESP, which, respectively, had or did not have classical N-terminal signal peptides. Figure 3a, b shows Venn diagrams for the sets of genes encoding “classical” (Figure 3a) and “non-classical” (Figure 3b) ESP, respectively, which were included in the molecular signatures of body parts. Approximately 35% (297/852) of the genes encoding “classical” ESP had a noticeable expression level in all body parts considered. At the same time, more than half (143/282) of genes encoding “non-classical” ESP presented this expression pattern. In both cases, the externa had the largest number of “specific” ESP: 325 and 56 “classical” and “non-classical” ESP, respectively. Dozens of ESP (101 “classical” and 35 “non-classical”) were common for the three interna parts considered.\n\n(a, b) Venn diagram for sets of potential “classical” (a) and “non-classical” (b) ESP encoded by genes from molecular signatures of the body parts. (c) Clouds of parental bioprocesses enriched by genes encoding “classical” ESP. Most often the bioprocess was found in the list of enriched bioprocesses, the larger the word size. (d–e) Scanning electron microscope (SEM) photos of the interna of P. reticulata, 1 - rootlets; 2 – host tissues enlacing rootlets. (f) Confocal laser scanning microscopy (CLSM) photo of the interna of P. reticulata, scale bar 100µm, 3 - host tissues stained with antibodies against α-tubulin. Abbreviations: class/nonclassES – “classical” and “non-classical” ESP, respectively.\n\nBoth “classical” and “non-classical” ESP were divided into families based on their sequence similarity. For “classical” ESP, 35 families contained two to four proteins, while only two “non-classical” ESP were combined into one family. Most (579/852) of the “classical” ESP matched the MetazSecKB database, the hits being, e.g., mannose-binding proteins, serine proteinase, and cuticle proteins (Table S5, Extended data71). Only 58 “non-classical” ESP matched MetazSecKB, of which 35 were “uncharacterized proteins” (Table S5, Extended data71). All ESP were also compared to the NeuroPep database, with which only 14 “classical” ESP had hits (Table S5, Extended data71). The latter included cerebellin-1, kininogen-1, insulin-like growth factors I and II, neuroparsin-A, nucleobindin-2, and five representatives of the serpin family.\n\nFigure 3c shows bioprocesses enriched with genes encoding “classical” ESP. Among the body-parts-specific bioprocesses were the “molting cycle” and “chitin-based cuticle development” in the externa, “positive regulation of cell communication” and “muscle organ development” in the growing interna part, “immune response” and “regulation of apoptotic signaling pathway” in the main trunk and the thoracic part of interna, respectively. The majority (21/34) of enriched bioprocesses were common for two or more body parts considered. For example, “regulated exocytosis” (externa and growing part), “mesoderm development” (the growing part and the main trunk), “cell recognition” (same), “proteolysis” (all body parts considered), “motor neuron axon guidance” (same), “cell adhesion” (same), and “neuron recognition” (externa and thoracic part of interna). The activity of the bioprocesses associated with the involvement of the nervous system is consistent with the fact that trophic rootlets of P. reticulata were enlaced by a network of host's neurons marked by a presence of a-tubulin and serotonin (Figure 3d–f).\n\nAll ESP analysis results are presented in Table S5 (Extended data71).\n\nAlmost all (12,618 / 12,620) P. reticulata genes were distributed across 17 phylostrata, i.e. sets of genes that coalesce to founder genes having a common phylogenetic origin72 (Table S6, Extended data) The three largest phylostrata were “Cellular organisms” (32.34%, 4,082 genes), “Eukaryota” (22.25%, 2,809 genes), and species-specific ones (13.7%, 1,729 genes) (Figure 4a). Less than 100 genes were assigned to “Ecdysozoa” (50 genes), “Panarthropoda”44, “Mandibulata” (63 genes), “Crustacea”28, and “Hexanauplia”6. Phylostratum “Cirripedia”, which included two barnacles, A. amphitrite and P. reticulata, consisted of 363 genes.\n\n(a) Phylostratigraphic composition analysis results for P. reticulata reference gene set (“reference”), set of genes with noticeable (≥ 2 transcripts-per-million) expression in all female body parts considered (“common expr”), sets of genes over-expressed (externa/growing/main trunk/thoracic_overexpr) and sets of genes encoding potential “classical” (“classical_ESP”) and “non-classical” (“nonclassical_ESP”) excretory/secretory proteins (ESP). (b, c) Relative mean expression levels of phylostrata which occurred “before” (b) or “after” (c) division of Crustacea. (d) Transcriptome Age Indices (TAI) variation for female body parts considered. A lower TAI value describes an “older” transcriptome, whereas a higher TAI denotes a “younger” one. (e) The cumulative phylostrata contribution to the final (global) TAI profile.\n\nThe phylostratigraphy results were also used for composition analysis of various gene sets (Figure 4a). More than half of the genes with expression ≥ 2 TPM in all body parts considered belonged to “Cellular organisms” and “Eukaryota” phylostrata. The proportion of species-specific genes in this set was approximately 7% (490/6,829). Noticeable differences in the contributions of different phylostrata to the sets of over-expressed genes were found. For example, approximately 31% (88/283) of such genes in the thoracic interna part belonged to the species-specific phylostratum. In contrast, in other parts of the body, the proportion of species-specific genes from the total number of over-expressed genes did not exceed 16%. A complex phylostratigraphic composition was also revealed for genes encoding both \"classical\" and \"non-classical\" ESP. “Cellular organisms” phylostratum made the greatest contribution to the “classical” ESP (32.16%), while the species-specific phylostratum contributed most to the “non-classical” ESP (38.3%).\n\nWe divided the phylostrata into two groups: before the divergence of Crustacea (from “Cellular organisms” to “Crustacea”) and after this event (from “Multicrustacea” to “P. reticulata”). The relative expression patterns of the phylostrata are shown in Figure 4b, c. All phylostrata except the species-specific one had the highest relative expression in the externa, while the highest expression of the species-specific phylostratum was recorded in the thoracic part of the interna. At the same time, 14 out of 17 phylostrata had the least expression in the main trunk of the interna, the remaining three being “Bilateria”, “Pancrustacea”, and “Multicrustacea”.\n\nOne metric to quantify transcriptome conservation on a global scale is the TAI73, which denotes the average transcriptome age throughout the biological process of interest64. The TAI was measured for each part of the female P. reticulata body. The higher the value of the TAI, the greater the contribution of the “young” phylostrata. Significant differences between the TAI of different body parts were revealed. The TAI of the thoracic part of the interna was the highest (4.33), whereas the TAI of the growing part of the interna was the lowest (4.21) (Figure 4d). The partial contribution of the “P. reticulata” phylostratum to the TAI of the body part was about approximately three times greater than the contribution of any other phylostratum (Figure 4e).\n\nWe extracted the top-500 with the largest contribution to the TAI of body parts from the genes with the GO annotation and performed GSEA for these gene sets (Figure 5). Among the “specific” bioprocesses were “embryonic organ development” and “male sex differentiation” in the externa, “cell fate commitment involved in the formation of primary germ layer” and “positive regulation of chemotaxis” in the growing part of the interna, “formation of primary germ layer” and “gland morphogenesis” in the main trunk of the interna, and “response to external stimulus” and “cell population proliferation” in its thoracic part. Only four bioprocesses were common for all the female body parts considered: “stem cell population maintenance”, “regulation of anatomical structure morphogenesis”, “chitin-based cuticle development”, and “animal organ morphogenesis”. Developmental processes were registered in each of the female body parts studied (Figure 5).\n\nClouds of parental bioprocesses enriched with top-500 genes with both Gene Ontology (GO) annotation and large contribution to TAI of the body part under consideration. The more often the bioprocess was found in the list of enriched bioprocesses, the larger the word size.\n\nPhylostratigraphy and evolutionary transcriptomics results are presented in Tables S6 and S7 (Extended data74,75), respectively.\n\n\nDiscussion\n\nIn this study, we obtained the first transcriptomes of a rhizocephalan and made a comparative analysis of different body parts of an adult female rhizocephalan. Our results are a step towards understanding the functioning of these highly specialized parasites and the trends of their evolutionary history. Below we discuss 1) how the molecular signatures helped us to verify the functional role of each part of the parasite’s body; 2) potential excretory/secretory proteins and their putative role in host-parasite interactions; 3) the trends of rhizocephalan evolution derived from the phylostratigraphy and evolutionary transcriptomics results.\n\nWe consider contamination identification and elimination in molecular biological studies of non-model parasitic organisms as one of the main challenging tasks. In our case, this challenge was aggravated by the fact that both the parasite and its host were crustaceans. This means that approaches based on database comparison could be less effective than usual at separating the reads from different sources. For this reason, we chose an MCSC algorithm, which classifies sequences based on the analysis of their properties. Based on the results of a preliminary orthogroup reconstruction (data not shown) and the reconstruction after decontamination and considering that there was an approximate 14% reduction in the number of duplicates of single-copy metazoan orthologues, we can assume that at least part of the signal from the host was removed. In further analyses, we focused on 12,620 protein-coding genes with a noticeable expression level. The results indicate that the reference gene set obtained in our study corresponds to those of other crustacean species in quality and completeness. The results of the analysis of orthogroups revealed that the proteome of P. reticulata was more similar to that of Amphibalanus amphitrite than to any other crustacean species involved in our study. Amphibalanus amphitrite belongs to Thoracica, a sister group of Rhizocephala. Thoracica barnacles are mostly free-living but also highly transformed crustaceans. A comparative analysis involving numerous representatives of these two sister taxa may uncover evolutionarily conservative mechanisms of transformation of adult rhizocephalans.\n\nThe body of a female rhizocephalan is divided into the externa and an extensive interna, which has different ultrastructural organization in different parts (76 and Miroliubov unpublished data). Our aim was to make a detailed record of the genome’s activity in different parts of the female body. Therefore, we used the soft threshold of 2 TPM as a condition for identifying genes whose expression contributed to the molecular signature of the sample under consideration. The results of the study of gene expression in different parts of the female body showed that: 1) slightly more than half of the identified protein-coding genes worked in all the examined parts of the body, 2) the lists of genes with an increased expression differed greatly between body parts, 3) the externa always clustered separately from the interna, although the differences were also found between the sites of the interna. These results suggest that the morphological heterogeneity of the female body is reflected in the spatial differences of gene expression (molecular heterogeneity).\n\nHowever, we suggest the molecular signature of the body part may be considered as a derivative of the transcriptomes of all cell types included in the body region analyzed. It also depends on the organism’s response to various stimuli and conditions (for example, the host's immune response, O2 level and concentrations of different metabolites in the host’s hemolymph)15,77. Therefore, the more similar the cellular composition of body parts or the set of factors affecting them are, the more similar their molecular signatures will be. In our study all parts of the interna clustered together and were separate from the externa cluster. At the same time, the differences of the biological replicates of the externae could be explained by the fact that the externa contains embryos at different stages of development. The signal from the embryos was probably so strong that even the pooling of samples did not smooth out the differences.\n\nOur results indicate that various processes are at work in different body parts of female rhizocephalan. Active developmental processes were registered in the externa, which could be expected considering that it contains numerous embryos, while active metabolism processes were recorded in the interna (Figure 2c). These results are consistent with the classical concepts of the functional role of individual parts of the rhizocephalan body1,2.\n\nHowever, some of our findings are at odds with the classical views. Previous morphological studies have postulated that the ovary is located in the visceral mass of the externa2. However, the GSEA revealed that the female germ cells formed in the interna. Moreover, in histological sections we observed some cells floating in the central lumen of the main trunk, that looked like primary germ cells. Such cells have been described before78,79 but the authors assumed them to be stem cells, responsible for the formation of new buds of externae. Based on histological data and the GSEA results, we suggest that these cells are more likely to be female germ cells. We suppose that female germ cells begin and/or continue to form in the interna and then migrate to the externa, where they mature and are fertilized. In our opinion, the ovary of rhizocephalans is diffused within the interna, while the externa serves merely as a brooding chamber.\n\nOur finding of a “diffused” ovary prompts a reconsideration of the phenomenon of rhizocephalan “coloniality”. As noted above, some rhizocephalans form numerous externae. It has been suggested that each externa is a separate reproductive module, and the entire animal has been considered as a colony2,78. However, if the ovary is in fact diffused and scattered across the interna and if numerous externae are merely brooding chambers, the term “coloniality” does not seem suitable for Rhizocephala. This issue calls for further research with the use of molecular and morphological methods.\n\nRegardless of whether a rhizocephalan barnacle is a colony or an individual organism, it has to communicate with the host via special excretory molecules involved in particular bioprocesses3. We found that the composition of the potential excretome/secretome varied in different parts of the female rhizocephalan body and showed the character of the distribution of the secreted substances. For instance, in the externa there seems to be an active excretion/secretion of proteins involved in the storage of nutrients in the developing embryos as well as those involved in the molting cycle. At the same time, proteins responsible for muscle development were also among of the potential excretome/secretome of the growing part of the interna. These data confirm that the muscular system is formed in a growing tip of the main trunk76.\n\nPotential excretory/secretory proteins involved in neuron axon guidance were found in both the externa and the interna. While neuron axon guidance in the externa is probably associated with the offspring development, the evidence of this process in the interna is less expected and more interesting. A direct contact between the parasite and the host’s nervous system was shown in this study and in our previous research3. We can expect the parasite to emit attracting signals, directing the host’s nervous system towards and along the interna. In addition, the excretory proteins involved in cell adhesion could also play an important role in the formation of a neural network around the rootlets. However, it cannot be ruled out that this transcriptome signal comes from the host tissues surrounding the rootlets of the parasite, since it is technically impossible to completely separate interna from the host’s tissues. Nevertheless, an intimate host-parasite interaction is an intriguing phenomenon requiring in-depth research.\n\nThe evolution of parasitic barnacles and their interactions with the hosts remains enigmatic80. In particular, this concerns the molecular basis of both the formation of phenotypes at different stages of the rhizocephalan life cycle and the interaction of the parasite with the host. At the same time, the principle of genomic phylostratigraphy implies that the genome of every extant species retains parts of the picture of the evolutionary epochs72. In order to determine how the P. reticulata gene set changed during evolution, we applied modern implementations of the phylostratigraphy to identify of gene groups with a common phylogenetic origin, called “phylostratum” or “phylostrata” in the plural form. The results of the phylostratigraphy analysis indicate that almost all P. reticulata genes were successfully distributed into 17 phylostrata. The third largest phylostratum was a species-specific one, which also probably includes genus- and family-specific orthologs. Given the transcriptome obtained in our study is the first reported rhizocephalan transcriptome, it is difficult to determine what percentage of proteins from this phylostratum belongs to each of these categories. Nevertheless, we are confident that a more detailed analysis of this particular phylostratum will allow us to identify the molecular basis of the Rhizocephala-specific biological traits. However, it should be kept in mind that genes need a cellular environment, the combined action of multiple other genes, as well as certain conditions to have an observable effect on organisms81.\n\nIt should be noted that we analysed the reference gene set reconstructed based on the transcriptomic data, and the transcripts of poorly expressed or completely silent genes may be absent from our data. However, since we obtained transcriptomes of different regions of the female body and used a soft expression level threshold, we may be fairly sure that many genes active in the adult female were represented in the reference gene set.\n\nOur results indicate that evolutionarily younger genes make a relatively large contribution to the signatures of the externa and the thoracic part of the interna. The transcriptional “youth” of the externa could be associated with the fact that the samples contained highly modified males2. Our assumption is also based on the fact that we observed \"male sex differentiation\" among the lists of bioprocesses enriched by genes with the largest contributions to the TAI of externa. On the contrary, the signature of the thoracic part probably does not have any additional components. That is, we can assume that a high TAI value for this region may be associated with the evolutionary transformation of the region itself. We have already previously identified morphological heterogeneity of the entire body of an adult rhizocephalan female9,76. Given the differences between body regions in morphology, we also expected to find molecular and functional heterogeneity in the body of an adult female P. reticulata. The results obtained from the studies indicate that such heterogeneity manifests itself not only in the expression of individual genes and the activity of bioprocesses, but also in the contributions of various phylostrata to the molecular signatures of interna regions studied. Further research should be directed towards a more detailed study of the identified differences in TAI between interna regions. At the same time, we assume that, for example, the revealed transcriptional “oldness” of the growing interna region is presumably due to the activity of conservative processes, including those associated with the cell cycle.\n\nThe GSEA results for genes with the greatest contribution to TAI, both in the interna and in the externa, revealed many developmental processes. One may get the impression that rhizocephalans have an incomplete and endless metamorphosis. Taking into account that the adult female body originates from a fraction of the larval body, we are inclined to agree with the hypothesis suggested by Glenner and Høeg80. It postulates that ancestors of rhizocephalans were filter-feeding epibiotic barnacles and the interna of an adult female originates from the part of the larval body homologous to the peduncle of a Goose barnacle, whose metamorphosis went the “wrong” way. The peduncle separated from the rest of the body and gave rise to the interna80,82. Nevertheless, a contemporary species can only serve as a proxy for an ancestral model. More transcriptomic/genomic data on other rhizocephalans are necessary for a reliable reconstruction of the evolutionary history of this unique group of parasites.\n\nTo sum up, the first comparative transcriptomic results for rhizocephalans brought us closer to understanding molecular mechanisms underlying the biology of these amazing parasites. We established the molecular and functional heterogeneity of the female rizocephalan body in addition to the known morphological one. A similarity was found between the set of protein-coding genes of the P. reticulata and that of a free-living representative of the sister taxon. Both bioinformatic data analysis and histological results indicated the presence of germ cells in the lumen of the interna, which casts doubt on the rhizocephalan coloniality. The molecular basis of the interaction between the nervous system of the host and the parasite's interna was determined. We established the differences between body parts in terms of phylostrata expression and their contribution to molecular signatures. Our results could indicate that rhizocephalans “got stuck in the metamorphosis” even in their reproductive stage. Our study can serve as a basis for further research on rhizocephalan evolution.\n\n\nData availability\n\nNCBI BioProject: Peltogaster reticulata Raw sequence reads, accession number: PRJNA798055.\n\nFigshare: Table S1. Summary of paired-end read libraries preparation results, https://doi.org/10.6084/m9.figshare.1930748666\n\nFigshare: Table S2. Peltogaster reticulata reference sequence set annotation results, https://doi.org/10.6084/m9.figshare.1930751667\n\nFigshare: Table S3. Gene expression quantification and analysis results, https://doi.org/10.6084/m9.figshare.1930754969\n\nFigshare: Table S4. Gene Set Enrichment Analysis (GSEA) results for the molecular signatures and sets of over-expressed genes, https://doi.org/10.6084/m9.figshare.1930757070\n\nFigshare: Table S5. Potential excretory/secretory proteins analysis results, https://doi.org/10.6084/m9.figshare.1930759471\n\nFigshare: Table S6. Phylostratigraphic affiliation analysis results for different set of sequences, https://doi.org/10.6084/m9.figshare.1930760674\n\nFigshare: Table S7. Evolutionary transcriptomics results, https://doi.org/10.6084/m9.figshare.1930762175\n\nFigshare: Figure S1. Phylogenetic relationships between the crustacean species based on orthologs analysis results, https://doi.org/10.6084/m9.figshare.1930744468",
"appendix": "Acknowledgements\n\nWe thank the staff of the Resource Centers “Microscopy and Microanalysis”, “Molecular and Cell Technologies”, and “The Bio-Bank” of the Research Park of St Petersburg State University and “Taxon” Research Resource Center (http://www.ckp-rf.ru/ckp/3038/) of the Zoological Institute of the Russian Academy of Sciences for technical assistance. Data analysis was performed at the Bioinformatics Shared Access Center of the Institute of Cytology and Genetics of the Siberian Branch of the Russian Academy of Sciences. We are grateful to Dr. Olga Korn and the diving team of the Marine Biological Station “Vostok” of National Scientific Center of Marine Biology, Far East Branch of Russian Academy of Science, for the help with the collection of specimens. We are grateful to Dr. Igor Adameyko and Natalia Lentsman for help with the manuscript preparation. 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PubMed Abstract | Publisher Full Text\n\nEmanuelsson O, Nielsen H, Brunak S, et al.: Predicting subcellular localization of proteins based on their N-terminal amino acid sequence. J Mol Biol. 2000; 300(4): 1005–16. PubMed Abstract | Publisher Full Text\n\nKrogh A, Larsson B, von Heijne G, et al.: Predicting Transmembrane Protein Topology with a Hidden Markov Model: Application to Complete Genomes. J Mol Biol. 2001; 305(3): 567–80. PubMed Abstract | Publisher Full Text\n\nMiele V, Penel S, Duret L: Ultra-fast sequence clustering from similarity networks with SiLiX. BMC Bioinformatics. 2011; 12(1): 116. PubMed Abstract | Publisher Full Text | Free Full Text\n\nWang Y, Wang M, Yin S, et al.: NeuroPep: A comprehensive resource of neuropeptides. Database (Oxford). 2015; 2015: bav038. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMeinken J, Walker G, Cooper CR, et al.: MetazSecKB: The human and animal secretome and subcellular proteome knowledgebase. Database (Oxford). 2015; 2015: bav077. PubMed Abstract | Publisher Full Text | Free Full Text\n\nArendsee Z, Li J, Singh U, et al.: Phylostratr: A framework for phylostratigraphy. Bioinformatics. 2019; 35(19): 3617–27. PubMed Abstract | Publisher Full Text\n\nDrost HG, Gabel A, Liu J, et al.: MyTAI: Evolutionary transcriptomics with R. Bioinformatics. 2018; 34(9): 1589–90. PubMed Abstract | Publisher Full Text | Free Full Text\n\nSchindelin J, Arganda-Carreras I, Frise E, et al.: Fiji: an open-source platform for biological-image analysis. Nat Methods. 2012; 9(7): 676–82. PubMed Abstract | Publisher Full Text | Free Full Text\n\nNesterenko M: Table S1. Summary of paired-end read libraries preparation results. figshare. Journal contribution, 2022. http://www.doi.org/10.6084/m9.figshare.19307486.v1\n\nNesterenko M: Table S2. Peltogaster reticulata reference sequence set annotation results. figshare. Journal contribution, 2022. http://www.doi.org/10.6084/m9.figshare.19307516.v1\n\nNesterenko M: Figure S1. Phylogenetic relationships between the crustacean species based on orthologs analysis results. figshare. Figure, 2022. http://www.doi.org/10.6084/m9.figshare.19307444.v2\n\nNesterenko M: Table S3. Gene expression quantification and analysis results. figshare. Journal contribution, 2022. http://www.doi.org/10.6084/m9.figshare.19307549.v1\n\nNesterenko M: Table S4. Gene Set Enrichment Analysis (GSEA) results for the molecular signatures and sets of over-expressed genes. figshare. Journal contribution, 2022. http://www.doi.org/10.6084/m9.figshare.19307570.v1\n\nNesterenko M: Table S5. Potential excretory/secretory proteins analysis results. figshare. Journal contribution, 2022. http://www.doi.org/10.6084/m9.figshare.19307594.v1\n\nDomazet-Loso T, Brajković J, Tautz D: A phylostratigraphy approach to uncover the genomic history of major adaptations in metazoan lineages. Trends Genet. 2007; 23(11): 533–9. PubMed Abstract | Publisher Full Text\n\nDomazet-Lošo T, Tautz D: A phylogenetically based transcriptome age index mirrors ontogenetic divergence patterns. Nature. 2010; 468(7325): 815–8. PubMed Abstract | Publisher Full Text\n\nNesterenko M: Table S6. Phylostratigraphic affiliation analysis results for different set of sequences. figshare. Journal contribution, 2022. http://www.doi.org/10.6084/m9.figshare.19307606.v1\n\nNesterenko M: Table S7. Evolutionary transcriptomics results. figshare. Journal contribution, 2022. http://www.doi.org/10.6084/m9.figshare.19307621.v1\n\nMiroliubov AA: Muscular system in interna of Peltogaster paguri (Rhizocephala: Peltogastridae). Arthropod Struct Dev. 2017; 46(2): 230–5. PubMed Abstract | Publisher Full Text\n\nShirley SM, Shirley TC, Meyers TR: Hemolymph responses of Alaskan king crabs to rhizocephalan parasitism. Can J Zool. 1986; 64(8): 1774–81. Publisher Full Text\n\nShukalyuk A, Isaeva V, Kizilova E, et al.: Stem cells in the reproductive strategy of colonial rhizocephalan crustaceans (Crustacea: Cirripedia: Rhizocephala). Invertebr Reprod Dev. 2005; 48(1–3): 41–53. Publisher Full Text\n\nIsaeva VV, Shukalyuk AI, Trofimova AV, et al.: The structure of colonial interna in Sacculina polygenea (Crustacea: Cirripedia: Rhizocephala). Crustacean Research. 2001; 30: 133–46. Publisher Full Text\n\nGlenner H, Høeg JT: A Scenario for the Evolution of the Rhizocephala. In: Escobar-Briones E, Alvarez F, editors. Modern Approaches to the Study of Crustacea. Springer, Boston, MA; 2002; 301–10. Publisher Full Text\n\nOrgogozo V, Morizot B, Martin A: The differential view of genotype-phenotype relationships. Front Genet. 2015; 6: 179. PubMed Abstract | Publisher Full Text | Free Full Text\n\nGlenner H, Hebsgaard MB: Phylogeny and evolution of life history strategies of the Parasitic Barnacles (Crustacea, Cirripedia, Rhizocephala). Mol Phylogenet Evol. 2006; 41(3): 528–38. PubMed Abstract | Publisher Full Text"
}
|
[
{
"id": "139230",
"date": "27 Jun 2022",
"name": "Andreas Hejnol",
"expertise": [
"Reviewer Expertise comparative transcriptomics in development and evolution"
],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis study provides the first glimpse into the transcriptomics of a parasitic rhizocephalan crustacean. The paper reads well, its coherent structure is easy to follow. The language used is appropriate for a publication and words are mindfully used when interpreting the results.\nThe authors set out to provide transcriptomic data of the parasitic barnacle Peltogaster reticulata, a timely goal as this highly specialized group of crustaceans is heavily understudied. This, with the addition of the current study, appears to change as a novel chromosome level genome assembly has been recently made available for another rhizocephalan species (Sacculina carcini). In this study different regions of the organism were selected, sequenced and their transcriptomic data were contrasted to each other. I found this approach sound to decipher the different roles of specialized body regions. I missed the description of the criteria used for separating the different body regions, especially for the internal structures.\nThe authors successfully recognized and removed the challenge of biological contamination of the data. A thorough and careful preprocessing of the data was performed. The generation of a high-quality transcriptome assembly is pivotal for any project with similar aims. Therefore, the authors spend a tremendous amount of time assuring the quality of the assembly and annotation. I liked the attention to the details during these steps. With several filtering criteria, they arrive at a set of contigs which are plausible. From the quality assessment, I missed the report of the full BUSCO scores (if BUSCO scores are the reported scores), not just the single-copy ortholog and duplicate, but also fragmented and missing proportions. Furthermore, with such strong filtering steps, valid data is also discarded in parallel with noise. To alleviate this problem generally multiple assemblers with multiple k-mer sizes are utilized and redundancy across the assemblies is dealt with downstream of the assembly (eg. DOI: 10.7717/peerj.5428 or https://doi.org/10.111/1755-0998.13593).[ref-1],[ref-2] This approach in my experience will usually greatly improve the assemblies, an improvement which might marginally benefit the authors of this study.\nNext, the focus was shifted to quantifying the expression of genes in the body regions and finding differentially expressed genes among them. Gene ontological enrichment was performed on different sets of genes (molecular signature set, overexpressed set and different phylostratographic sets). The output of these analyses with the addition of histological data prompted the authors to hypothesize that female germ cells are formed in the internal trunk region and migrate from here to the externa part. Indeed, such results combined with previous reports hint towards this possibility, something which could be tested with an in situ hybridization for germ cell marker genes such as for example vasa or nanos (both of which are present in the assembled transcriptome).\nUsing various in silico methods the authors outlined a set of potential excretory/secretory proteins. Several uncharacterized proteins are retrieved from this identification method, some of which could potentially be involved at the interface between parasite and host, as suggested by the authors. Unfortunately, without further validation, this suggestion persists as a testable hypothesis.\nResults from the phylostratographic analyses emphasize further the unique biology of parasitic barnacles. The interpretation of the results by the authors was legitimate. My concern with such approaches is their sensitivity to the BLAST algorithm (see a thorough description of the issue here: https://drostlab.github.io/myTAI/articles/Phylostratigraphy.html)\nFurthermore, I found the wording used for describing the datasets utilized in similarity searches confusing. How many species were used for the phylostratographic mapping and how many of them were metazoan?\nA rich resource of results was provided by the authors. With the descriptions provided the additional data is easily interpretable. At the moment the analyses conducted in this study are irreproducible without the custom scripts used by the authors. These should be made available for the community who wishes to replicate the study or to further investigate these intriguing parasitic barnacles.\nOverall I found this publication exciting and serving as a springboard for future studies on parasitic barnacle biology or evolutionary biology of parasitic life histories. If the authors complement the manuscript with the missing parts outlined above (criteria used for separating the body regions, phylostratography wording, missing scripts) the article would be greatly enhanced.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Partly\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nPartly\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes",
"responses": [
{
"c_id": "9169",
"date": "09 Jan 2023",
"name": "Maksim Nesterenko",
"role": "Author Response",
"response": "We thank the reviewer for their very helpful, clear, and pertinent remarks. Each original comment is in italics and our responses are in bold. The authors set out to provide transcriptomic data of the parasitic barnacle Peltogaster reticulata, a timely goal as this highly specialized group of crustaceans is heavily understudied. This, with the addition of the current study, appears to change as a novel chromosome level genome assembly has been recently made available for another rhizocephalan species (Sacculina carcini). In this study different regions of the organism were selected, sequenced and their transcriptomic data were contrasted to each other. I found this approach sound to decipher the different roles of specialized body regions. I missed the description of the criteria used for separating the different body regions, especially for the internal structures. We have added a more defined description of the way of separating different body parts in the Methods section. The authors successfully recognized and removed the challenge of biological contamination of the data. A thorough and careful preprocessing of the data was performed. The generation of a high-quality transcriptome assembly is pivotal for any project with similar aims. Therefore, the authors spend a tremendous amount of time assuring the quality of the assembly and annotation. I liked the attention to the details during these steps. With several filtering criteria, they arrive at a set of contigs which are plausible. From the quality assessment, I missed the report of the full BUSCO scores (if BUSCO scores are the reported scores), not just the single-copy ortholog and duplicate, but also fragmented and missing proportions. We refined the results of the comparison against the database of single-copy orthologs by indicating the percentages of orthologs that were duplicated, fragmented, or missing from our dataset. For example: “The comparison with a single-copy metazoan orthologues database revealed that in the reference protein set of P. reticulata 75.6% of the orthologues were assembled completely (Single: 70.4%; Duplicated: 5.2%), whereas 3.4% and 21% of the sequences were fragmented or absent, respectively.” Furthermore, with such strong filtering steps, valid data is also discarded in parallel with noise. To alleviate this problem generally multiple assemblers with multiple k-mer sizes are utilized and redundancy across the assemblies is dealt with downstream of the assembly (eg. DOI: 10.7717/peerj.5428 or https://doi.org/10.111/1755-0998.13593).[ref-1],[ref-2] This approach in my experience will usually greatly improve the assemblies, an improvement which might marginally benefit the authors of this study. We fully agree that the proposed method can indeed improve assembling results. Moreover, using multiple assemblers and re-running programs with different k-mer lengths was considered as a possible strategy in the early stages of planning a general analysis scheme based on the results presented in the TransRate publication (DOI: 10.1101/gr.196469.115). However, for the initial assembly, we opted for the Trinity program, which shows excellent assembly results even with default parameters. Now we are starting work on a new article, which will include new data, with the help of which we plan to improve the assembly presented in the current manuscript and refine the results obtained on its basis. As part of the new study, we also plan to test the approach you indicated in order to obtain the most complete and high-quality assembly of the transcriptome. Next, the focus was shifted to quantifying the expression of genes in the body regions and finding differentially expressed genes among them. Gene ontological enrichment was performed on different sets of genes (molecular signature set, overexpressed set and different phylostratographic sets). The output of these analyses with the addition of histological data prompted the authors to hypothesize that female germ cells are formed in the internal trunk region and migrate from here to the externa part. Indeed, such results combined with previous reports hint towards this possibility, something which could be tested with an in situ hybridization for germ cell marker genes such as for example vasa or nanos (both of which are present in the assembled transcriptome). Soon we are going to test our hypothesis by an in situ hybridization for germ cell marker genes. However, we plan to publish the results obtained in a separate article. Using various in silico methods the authors outlined a set of potential excretory/secretory proteins. Several uncharacterized proteins are retrieved from this identification method, some of which could potentially be involved at the interface between parasite and host, as suggested by the authors. Unfortunately, without further validation, this suggestion persists as a testable hypothesis. We fully agree that the presented results are preliminary and are aimed primarily at narrowing the range of potential targets for a more detailed study. Now we are working on it with the use of a proteomic approach and hope to publish the results soon in our new publications. Results from the phylostratographic analyses emphasize further the unique biology of parasitic barnacles. The interpretation of the results by the authors was legitimate. My concern with such approaches is their sensitivity to the BLAST algorithm (see a thorough description of the issue here: https://drostlab.github.io/myTAI/articles/Phylostratigraphy.html) Before starting the phylostratigraphic analysis, we carefully reviewed various materials, including the ones you mentioned. So, for example, according to this publication (https://doi.org/10.1093/molbev/msw284) BLAST is an appropriate and sufficiently sensitive tool in phylostratigraphic analysis that does not appear to introduce significant biases into evolutionary pattern inferences. Nevertheless, we agree that more complex comparisons of different methods are required to determine how correctly they detect distant homologs. By the way, as one of the promising directions for improving the strategy for phylostrata identification the Leapfrog method (doi:10.1101/gr.216226.116) proposed by your scientific group and based on the inclusion of «bridge» species can be considered. Furthermore, I found the wording used for describing the datasets utilized in similarity searches confusing. How many species were used for the phylostratographic mapping and how many of them were metazoan? In total, in addition to the P. reticulata, 139 species were included in the analysis, of which 44 were representatives of the Metazoa. We not only have provided this information in the Methods but have also included a phylogenetic tree of all species considered in the Supplementary materials. Thank you very much for pointing out to us that we missed this important information. A rich resource of results was provided by the authors. With the descriptions provided the additional data is easily interpretable. At the moment the analyses conducted in this study are irreproducible without the custom scripts used by the authors. These should be made available for the community who wishes to replicate the study or to further investigate these intriguing parasitic barnacles. All custom Python and R scripts used in the study are publicly available: https://github.com/maxnest/From_head_to_rootlet"
}
]
},
{
"id": "153684",
"date": "14 Nov 2022",
"name": "Viatcheslav Ivanenko",
"expertise": [
"Reviewer Expertise Morphology",
"development",
"phylogeny and ecology of symbiotic crustaceans."
],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 a completed work with new and significant scientific results. In my opinion, the manuscript is well written and illustrated, and the interpretation of the results raises no questions or doubts. The main remark concerns the fact that despite the uniqueness of many features of the biology and morphology of rhizocephalans, they are not the only highly modified crustaceans that parasitize crustaceans. In the first sentences of the manuscript or discussion, it would be appropriate to mention at least some poorly studied and highly modified copepods with root-like mouthparts that also parasitize other crustaceans. These copepods are belonging to the family Nicothoidae Dana, 1852-1853 and the synonymous family Choniostomatidae Hansen, 1886 (see Boxshall and Harrison, 19881).\nSome phrases may not be very well written, but I am not a native English speaker to make edits.\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nI cannot comment. A qualified statistician is required.\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes",
"responses": [
{
"c_id": "9170",
"date": "09 Jan 2023",
"name": "Maksim Nesterenko",
"role": "Author Response",
"response": "We thank the reviewer for their comments, which are listed below (in italics) with our responses (in bold): The main remark concerns the fact that despite the uniqueness of many features of the biology and morphology of rhizocephalans, they are not the only highly modified crustaceans that parasitize crustaceans. In the first sentences of the manuscript or discussion, it would be appropriate to mention at least some poorly studied and highly modified copepods with root-like mouthparts that also parasitize other crustaceans. These copepods are belonging to the family Nicothoidae Dana, 1852-1853 and the synonymous family Choniostomatidae Hansen, 1886 (see Boxshall and Harrison, 19881). You are absolutely correct in pointing out that rhizocephalans are not the only ones showing amazing body transformation among Crustacea. We have added information about parasitic copepods at the end of the second paragraph of the discussion. Some phrases may not be very well written, but I am not a native English speaker to make edits. Even though for both authors of the article English is not their native language, we have tried to do our best to convey our thoughts as correctly and clearly as possible. However, we have tried to improve some of the wording."
}
]
}
] | 1
|
https://f1000research.com/articles/11-583
|
https://f1000research.com/articles/12-25/v1
|
09 Jan 23
|
{
"type": "Research Article",
"title": "Attitudes of medical students towards the ethical and legal aspects of abortion: a cross-sectional study from Saudi Arabia",
"authors": [
"Fatima I. Alhumaid",
"Najyah A. Almohammedhusen",
"Nada A. AlMohammedsalem",
"Zainab A. Busbaih",
"Ritesh G. Menezes",
"Fatima I. Alhumaid",
"Najyah A. Almohammedhusen",
"Zainab A. Busbaih",
"Ritesh G. Menezes"
],
"abstract": "Abortion is defined as the termination of pregnancy which is a crucial issue to be addressed by multiple regulating systems such as health care providers and policy makers. The main aim of the study was to assess the attitudes of medical students in Imam Abdulrahman bin Faisal university towards abortion in different circumstances. This is a descriptive cross-sectional study in which a 20-items online questionnaire was distributed through social media platforms to medical students. The results of the study showed that the majority of the students believe abortion should be determined by law, religion and spousal consent. The majority as well support abortion in cases of endangered mother’s life, fetal life compromise and rape victims. However, they were against abortion in cases of financial incapacity of the parents and cases of unplanned pregnancy. The results of the study can be applied to improve medical education of abortion. More studies in this field of research are recommended for the purpose of providing more inclusive assessment of abortion attitudes in different medical education settings.",
"keywords": [
"Abortion",
"attitudes",
"legislation",
"medical students",
"Saudi Arabia"
],
"content": "Introduction\n\nAbortion is defined as the expulsion of a fetus from the uterus which results in termination of pregnancy. It can occur either spontaneously or intentionally. Spontaneous abortion occurs due to maternal or fetal diseases. On the other hand, abortion can be carried out intentionally for various reasons, such as medical conditions jeopardizing the mother’s life, or the fetus being diagnosed with a life-threatening illness. In this situation, it is called an induced abortion. A wide range of medical and surgical interventions can be implemented to induce abortion, depending on the gestational age, and the medical status of the mother and fetus. Abortion induction techniques include endometrial aspiration, dilatation and curettage, evacuation, injection, or ingestion of medical substances inducing uterine contractions.\n\nThe accessibility of induced abortion constitutes a web of legal, social, and cultural issues, with distinct legislation and limitations in different countries. This ranges from countries which broadly allow abortion to countries with restrictive abortion laws. Statistically, each year approximately 68,000 women die from unsafe abortion procedures. The lack of access to safe and legal abortion is a public health concern, as one of the most prominent contributors to maternal morbidity and mortality.1 Hemorrhage, infections, sepsis, and related complications are the most common attributing factors leading to death following unsafe abortion which consequently increases the mortality rates resulting from unsafe abortion. In addition, infertility is a long-term complication that arises following the induction of unsafe abortion.2 Hence, it is essential to ensure that every woman has the right to access legal and safe abortion services.\n\nDoctors are on the frontline as health care providers, and assessment of the medical and legal knowledge of doctors regarding abortion can positively affect the number of disadvantaged cases. Medical students are future doctors and therefore it is essential to learn about their attitudes towards abortion, a topic with profound ethical and legal implications. More regional studies in this area of research assessing different attitudes in different cultures are needed. The main purpose of this study was to assess the attitudes of medical students at a university in Saudi Arabia towards abortion in different circumstances, aiming to attain an overview of their current level of knowledge and awareness regarding ethical and legal aspects of abortion.\n\n\nMethods\n\nThis is a descriptive cross-sectional questionnaire-based study that was conducted by distributing an electronic survey among medical students at Imam Abdulrahman Bin Faisal University in Saudi Arabia. The target population was male and female medical students aged 18 and above who were enrolled at the university and were willing to participate. 1227 medical students were studying at the College of Medicine of the Imam Abdulrahman Bin Faisal University at the time when the study was conducted. The sample size was calculated using the population proportion-sample size calculator. A sample size of 293 was estimated with a 95% confidence level and 5% marginal error. Ethical approval was obtained from the Institutional Review Board (IRB) of the university (IRB-UGS-2021-01-398; 03-November-2021). Information pertaining to the introduction and objectives of the present study was provided to the participants on the first page of the online questionnaire survey and if they proceeded to complete the questionnaire, it was considered as the participants’ consent for participation. Moreover, student participation was anonymous and voluntary with the purpose of respecting their right of choice to participate in the present study.\n\nAn online self-administered questionnaire was developed based on previously published studies.3,4 This questionnaire was reviewed by two experts with experience in research methodology and knowledge on this specific research topic. The questionnaire was also piloted on a sample of 20 students and based on their comments the questionnaire was finalized. The finalized questionnaire was distributed through social media platforms to all medical students at the university during the months of November and December in 2021. The response rate was 25%.\n\nThe questionnaire contained 20 items categorized into four sections. The first section included student demographic data (age, sex, year of study, and marital status). In the second section, the participants were asked to evaluate eight statements related to the general support of abortion. An additional seven statements in the third section were related to abortion decision support in specific conditions based on their opinion, asking whether they strongly agreed/agreed/disagreed/strongly disagreed/were neutral. Finally, in the fourth section, students’ attitudes toward abortion were assessed through five different clinical circumstances, to evaluate their intentions to act based on their attitudes.\n\nData were analyzed using Statistical Package for the Social Sciences (SPSS) version 26 (IBM Corp., Armonk, New York). Normality was assessed by the Shapiro-Wilk Test. Descriptive statistics were used to report frequencies and proportions for the categorical responses. The association between categorical variables was checked using the Chi-square test. A p-value of <0.05 was considered significant in all cases. All the underlying data for the present study is available without restriction.\n\n\nResults\n\nA total of 308 participants took part in our study (Table 1). The mean age was 21 years, with female participants constituting over two-thirds of the population (70.1%). The majority of the population was unmarried (91.5%). Approximately one-third of the medical students were in the sixth year (36.8%). Second and third-year students were categorized as pre-clinical year students (38.1%) while fourth to sixth year students were categorized as clinical year students (61.9%).\n\nParticipant responses regarding general support for abortion provision are detailed in Table 2. Almost two-thirds of students believed that abortion did not fall in the category of murder (n=192, 62.5%). This belief was more common in female students than in males (66.6% vs 52.2% respectively; p=0.03). A similar proportion of single and married participants believed that abortion should not be considered murder (62.6% vs 61.5%; p=0.99). Furthermore, a significantly greater number of clinical year students, compared with pre-clinical students, also believed abortion did not fall in the category of murder (72.6% clinical and 46.2% pre-clinical; p<0.001).\n\nOver half of the participants believed that abortion decisions should not be made individually by the mother herself (n=161, 52.4%). This belief was more common amongst male medical students than female (70.4% vs 45.2% respectively; p <0.01). There was no significant difference in responses between married and unmarried individuals (61.5% vs 52%; p=0.63).\n\nOverall, more than 50% of the students believed that medical education provides enough information regarding ethical issues surrounding abortion (n=158, 51.4%). The proportion of female students (n=118, 53.8%) and male students (n=40, 45.4%) with this belief was similar (p=0.06). Although a numerically higher proportion of married participants believed this, the difference between married and single individuals was not statistically significant (53.3% vs 30.7% respectively; p=0.17).\n\nAbout half of the medical students asserted that medical education provided sufficient information regarding ethical issues surrounding abortion (n=158, 51.4%). A similar number of pre-clinical year and clinical year students were found to believe this (53.8% and 50% respectively; p=0.72).\n\nThe overall response of the sample population towards conditional support of provision of abortion is detailed in Table 3. When asked whether abortion should be provided in case of congenital anomalies not compatible with life, the majority of participants responded in favor of the provision (n=248, 80.8%). Female medical students were more likely to be in favor of abortion in this scenario (85.8% vs 68.1% respectively; p=0.002). The proportion of single and married participants with this belief was similar (80.8% vs 80.7% respectively; p=0.86). More than half of participants felt that abortion should not be provided to parents who are not financially capable (n=216, 70.4%;). Male medical students were more likely to believe this than female students (80.6% vs 66.2% respectively; p=0.07). A similar proportion of married and single participants also held this belief (80.7% vs 85.9% respectively; p=0.67).\n\nThe majority of the students were in favor of abortion being made available to rape victims (n=192, 62.5%), with female students significantly more likely to believe this than male students (68.9% vs 46.6% respectively; p=0.04). Furthermore, single participants (63.3%) were significantly more likely to be advocates of this belief than married individuals (53.8%; p<0.01).\n\nThe attitude of medical students towards abortion as future physicians was also assessed. The overall response is detailed in Table 4. More than two-thirds of the participants disagreed when asked if they would not perform an abortion at any cost (206, 67.1%). A nearly equal response in favor was observed between female and male medical students (69.8% vs 60.2% respectively; p=0.32). Marital status did not influence the response (single: 67.2% disagreed vs married: 61.5% disagreed; p=0.68).\n\nAbout half of the medical students preferred to advise the patient not to go through the procedure (n=154, 50.2%). Male students (59.1%) were more in favor of this than female students (46.5%; p=0.04). This response was similar between single and married students (50.5% vs 46.2% respectively; p=0.97).\n\nA significant number of participants disagreed with stopping other physicians from performing abortions (n=140,45.6%;). Female students (52.5%) were more in favor of this than male medical students (28.4%; p <0.01). There was no significant difference in responses between married and single participants (53.8% vs 44.8% respectively; p=0.56).\n\nThe independent-sample t-test to compare Likert scale score for attitude towards abortion as future physicians revealed no significant difference between male (n=14.9, SD=2.9) and female (n=13.8, SD=3.3) respondents (p=0.08).\n\n\nDiscussion\n\nIn the present study, we assessed various attitudes of medical students at a university in Saudi Arabia towards abortion in different circumstances.\n\nThe results of this study showed that 62.5% of the students believed that abortion did not fall in the category of murder, female students were more likely than males to hold this belief (p=0.03). In a comparable survey among Turkish university students, the percentage of male participants who agreed with the statement “abortion shouldn’t be allowed anyway” was higher than that of female participants, and the difference was statistically significant (p<0.001). In addition, Turkish students had a more conservative attitude toward abortion, with approximately 80% of the participants either having no opinion or being opposed to abortion, and 70% believing that eliminating a life is the only right of Allah.3 A similar conclusion was made in Jordan, where around 53% of students believed that abortion should be considered a form of murder.5 However, medical students’ attitudes toward abortion may change as they progress in their academic levels. In our study, most clinical-year students (72.6%) agreed that abortion isn’t considered a type of murder which was significantly higher than pre-clinical-year students (46.2%). Similar results were reported in a study that assessed midwifery students’ attitudes towards abortion in Poland. There was a significant difference between the first and the final year students’ attitudes toward abortion, where a higher number of the final year students were willing to participate in abortion under different circumstances than first-year students (71.9% vs 57.3%); their willingness to participate in abortion cases shows that they don’t believe that abortion is murder as well.6 Gleeson et al. encountered consistent results in their study, which found that second-year medical students in the United Kingdom were more pro-choice than first-year students.7 Similarly, significant differences in attitudes regarding abortion were observed between first- and fourth-year medical students in Papua New Guinea, indicating that acceptance of abortion grew with years of study.8\n\nMost of the students who participated in our study believed that abortion decisions should be determined by law and religion. This may reflect Saudi Arabia being an Islamic country that regulates most of its laws by the religion of Islam. Consequently, 74.3 % of the participants in the present study believed that abortion decisions should be determined by religion. A similar finding was reported in a systemic literature review of health care providers’ perceptions and attitudes towards abortion in sub-Saharan Africa and Southeast Asia. Most of the respondents of the studies included in the systemic review believe that only God can make decisions about life and death. Additionally, abortion was viewed as a sin.1 A study that assessed the attitudes towards abortion among the Muslim minority and non-Muslim majority in western Europe countries showed that Muslim’s negative attitude towards abortion was significantly influenced by religion.9\n\nIn addition to laws and religion, participants in the present study believed that spousal consent is required in making the abortion decision since 52.4% of them disagreed that a woman should have the right to decide for herself whether to have an abortion. This finding is inconsistent with the results of a study conducted among medical students in South Africa where most of the participants (70%) agreed that a woman should have the right to decide for herself whether to have an abortion or not.4 Similar results were found in a study conducted among physicians who provided clinical reproductive health care in the United States, where many participants strongly opposed that spousal notification (81.3%), and consent (86.6%) should be required for married women. Fewer strongly disagreed that parental notification (57.6%) and parental approval (66.9%) should be required for minors.10 Our findings suggest that male students in Saudi Arabia are more likely to disagree with the statement “abortion decision should be determined individually by the pregnant woman” than females and it is statistically significant. Similarly, male students were more likely to agree with the statement “spousal consent is essential to make abortion decisions” and this difference was also found to be statistically significant.\n\nMost of the students believed that abortion is warranted when a woman’s health is at risk, when serious fetal congenital defects are suspected, and in the case of rape. This is consistent with the results of other studies.4,5,11–16 Our study also showed that most of the participants were against permitting abortion in the case of financially incapable parents (70.3%), and in the case of unplanned pregnancy (82%).\n\nIn the present study, around 67% of the participants were against refusing to perform an abortion at any cost and 45.6% were against stopping other physicians from performing abortions. The percentage of participants in our study who agreed to refer patients to other physicians in situations where they would not perform abortion was much less than those reported in a South African study (47.2% vs 70%).4 However, a similar finding was concluded from a study assessing the attitudes of abortion objectors towards physician referral for abortion services in Colombia. Most of the moderate Colombian objectors felt comfortable referring patients to other physicians who would provide the required service for the patients as requested.17\n\nSanitya et al. discovered that health care providers’ positive attitudes about unplanned pregnancies and unsafe abortions increased dramatically after a training program to prevent unsafe abortion was implemented in Thailand.18 This emphasizes the value of training programs in raising awareness about unsafe abortion and providing safe abortion services. Overall, most participants (51.4%) in our study were satisfied with the current medical education regarding the ethical considerations surrounding abortion. Likewise, in a study from the United Kingdom, 57% of medical students believed that they received the appropriate amount of education on abortion.19 In contrast, a Malaysian study reported that more than 90% of medical students believed that there should be more education and training about the legal aspects of abortion, as well as increased knowledge about this.20\n\nThe number of studies showing the effect of abortion laws on women’s physical and mental health in the Middle East and North Africa is insufficient and there is no reliable data about unsafe abortion in this region.\n\nIn 2008, the World Health Organization reported that there were about 830,000 unsafe abortions in West Asia that caused 600 maternal deaths and 900,000 unsafe abortions in North Africa leading to 1500 annual maternal deaths.21 Therefore, abortion is considered a serious medical issue because of increased maternal mortality in cases of unsafe abortion.21 The results of the present study showed that the attitudes of medical students toward abortion are mostly positive, which is reassuring because medical students will play a major role in managing abortion and its complications in the future and raising awareness about such issues. Moreover, it is important to assess how future physicians will act toward abortion cases so that we can have an estimation of the possible ways of managing unsafe abortion cases in the future.\n\nA positive aspect of the present study is that it is one of the first studies to be conducted in Saudi Arabia to assess medical students’ attitudes towards abortion. An anonymous questionnaire was used to collect data. This made it suitable for the participants to possibly provide honest opinions about abortion, a very sensitive topic.\n\nHowever, the present study is not without limitations. Our study was conducted in a single center. Thus, the results of our study cannot be generalized to the entire medical student community in Saudi Arabia and aren’t applicable on a global scale. Moreover, this study included the current opinions of medical students which might change in the future as they qualify to practice as physicians. On the other hand, assessing physicians’ attitudes would provide a better understanding of abortion safety measures being considered. In addition, the study included more single students than married students and this might have led to an interpretive bias in results. Finally, the participants were asked to report their biological sex only. While social factors such as gender identification were not taken into account because it is not culturally an accepted phenomenon in Saudi Arabia.\n\n\nConclusions\n\nIn conclusion, 62.5% of the Saudi medical students who participated in this study believed that abortion is not a form of murder, with most of them holding the belief that abortion is warranted when a woman’s health is at risk, when serious fetal congenital defects are suspected, and in cases where pregnancy is a result of rape. In addition, most of the participants had a positive attitude toward abortion, and they believed that they have sufficient education about abortion, which is promising as abortion remains a debatable issue morally, ethically, and legally.\n\nFor future research\n\nA nationwide study involving the entire medical student community in Saudi Arabia is recommended for a broader picture of the attitudes of medical students, the future physicians, towards abortion. Such a study should be a multi-centered one with a larger sample size. Similar studies should be conducted to learn about the attitudes and practices of the present physicians toward abortion not just in Saudi Arabia, but in the rest of the Middle East as well.22\n\nFor policy and law makers\n\nThe laws of abortion are based on Islam. Thus, it is not amenable to changes. However, the policy and law makers need to provide clearer guidelines for abortion practices that can be easily accessible to those genuinely requiring these services in Saudi Arabia.\n\nFor medical students\n\nProvision of sufficient medical and legal education regarding abortion and the proper way to act in challenging situations is recommended, as unsafe abortion practices are known to increase maternal mortality. Lastly, raising the community’s awareness level of this sensitive topic via varied ways of communicating information such as public health campaigns with the involvement of medical students could be considered.",
"appendix": "Data availability\n\nDRYAD: Attitudes of medical students towards the ethical and legal aspects of abortion: a cross-sectional study from Saudi Arabia: https://doi.org/10.5061/dryad.dfn2z355s. 23\n\nThis project contains the following underlying data:\n\n• MedicaL_students_attitude_towards_abortion_last_version_.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\nReferences\n\nLoi UR, Gemzell-Danielsson K, Faxelid E, et al.: Health care providers’ perceptions of and attitudes towards induced abortions in Sub-Saharan Africa and Southeast Asia: systematic literature review of qualitative and quantitative data. BMC Public Health. 2015; 15: 139. Publisher Full Text\n\nWorld Health Organization: Unsafe Abortion: Global and Regional Estimates of Incidence of Unsafe Abortion and Associated Mortality. 6th ed.2008.\n\nFeyzal N, Polat G, Dasbas S: Opinions on abortion among a group of university students in Turkey. Int. J. Humanit. Soc. Sci. 2015; 5: 89–95.\n\nWheeler SB, Zullig LL, Reeve BB, et al.: Attitudes and intentions regarding abortion provision among medical school students in South Africa. Int. Perspect. Sex. Reprod. Health. 2012; 38: 154–163. PubMed Abstract | Publisher Full Text | Free Full Text\n\nSaadeh R, Alfaqih M, Odat A, et al.: Attitudes of medical and health sciences students towards abortion in Jordan. Biomed. Res. Int. 2021; 2021: 6624181.\n\nMichalik A, Zdun-Ryzewska A, Pieta B, et al.: Multicenter study on midwifery students’ attitudes towards abortion and it’s place in their future practice: comparison of respondents at early and late stages of the university education. Nurse Educ. Pract. 2019; 35: 42–47. PubMed Abstract | Publisher Full Text\n\nGleeson R, Forde E, Bates E, et al.: Medical students’ attitudes towards abortion: a UK study. J. Med. Ethics. 2008; 34: 783–787. PubMed Abstract | Publisher Full Text\n\nKolodziejczyk I, Kuzma J: Knowledge and attitudes towards abortion and euthanasia among health students in Papua New Guinea. Adv. Med. Educ. Pract. 2020; 11: 977–987. PubMed Abstract | Publisher Full Text | Free Full Text\n\nCarol S, Milewski N: Attitudes toward abortion among the Muslim minority and non-Muslim majority in cross-national perspective: can religiosity explain the differences? Sociol. Relig. 2017; 78: 456–491.\n\nDodge LE, Haider S, Hacker MR: Attitudes toward abortion among providers of reproductive health care. Womens Health Issues. 2016; 26: 511–516. PubMed Abstract | Publisher Full Text\n\nBaba CF, Casas L, Ramm A, et al.: Medical and midwifery students’ attitudes toward moral acceptability and legality of abortion, following decriminalization of abortion in Chile. Sex. Reprod. Healthc. 2020; 24: 100502. PubMed Abstract | Publisher Full Text\n\nFitzgerald JM, Krause KE, Yermak D: The first survey of attitudes of medical students in Ireland towards termination of pregnancy. J. Med. Ethics. 2014; 40: 710–713. PubMed Abstract | Publisher Full Text\n\nMatthews G, Atrio J, Fletcher H, et al.: Abortion attitudes, training, and experience among medical students in Jamaica, West Indies. Contracept. Reprod. Med. 2020; 5: 4. PubMed Abstract | Publisher Full Text | Free Full Text\n\nBuga GA: Attitudes of medical students to induced abortion. East Afr. Med. J. 2002; 79: 259–262. PubMed Abstract | Publisher Full Text\n\nNorris A, Bessett D, Steinberg JR, et al.: Abortion stigma: a reconceptualization of constituents, causes, and consequences. Womens Health Issues. 2011; 21: S49–S54. PubMed Abstract | Publisher Full Text\n\nKestler E: Obstetrician-gynecologists’ knowledge of and Attitudes toward medical abortion in Guatemala. Int. J. Gynecol. Obstet. 2012; 116: 120–123. PubMed Abstract | Publisher Full Text\n\nFink LR, Stanhope KK, Rochat RW, et al.: “The fetus is my patient, too”: attitudes toward abortion and referral among physician conscientious objectors in Bogota, Colombia. Int. Perspect. Sex. Reprod. Health. 2016; 42: 71–80. PubMed Abstract | Publisher Full Text\n\nSanitya R, Marshall A, Saengruang N, et al.: Healthcare providers’ knowledge and attitude towards abortions in Thailand: a pre-post evaluation of trainings on safe abortion. Int. J. Environ. Res. Public Health. 2020; 17: 3198. PubMed Abstract | Publisher Full Text | Free Full Text\n\nCohen P, Mayhew J, Gishen F, et al.: What should medical students be taught about abortion? an evaluation of student attitudes towards their abortion teaching and their future involvement in abortion care. BMC Med. Educ. 2021; 21: 4. PubMed Abstract | Publisher Full Text | Free Full Text\n\nTey N, Yew S, Low W, et al.: Medical students’ attitudes toward abortion education: Malaysian perspective. PLoS One. 2012; 7: e52116. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMaffi I, Tonnessen L: The limits of the law: abortion in the Middle East and North Africa. Health Hum. Rights. 2019; 21: 1–6. PubMed Abstract\n\nAlnamlah M, Itani S, Alqahtani M, et al.: Common medical ethics dilemmas: Few reflections from a Saudi perspective. J. Forensic Leg. Med. 2022; 90: 102394. Publisher Full Text\n\nAlhumaid FI, et al.:Attitudes of medical students towards the ethical and legal aspects of abortion: A cross-sectional study from Saudi Arabia, Dryad. [Dataset].2022. Publisher Full Text"
}
|
[
{
"id": "159557",
"date": "17 Feb 2023",
"name": "Lauren E Farmer",
"expertise": [
"Reviewer Expertise Medical student education in abortion",
"obstetrics and gynecology",
"contraception"
],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 think the author provides a nice study on the attitudes of students in their region. Here are some alterations/comments.\nAbstract:\n\nIn the abstract - abortion definition - please clarify that this induced abortion as opposed to other terms such as spontaneous abortion = miscarriage. Maybe be more specific.\n\nIn the abstract the majority of students believe abortion should be determined. Maybe state that the decision surrounding abortion should be determined by...\n\nHow can the results of the study be used to improve medical education of abortion? I would end with a stronger statement here\n\nComments on the introduction: Specify whether these abortions you're describing occur in the world vs. the region you're commenting on. Be more descriptive with the statistics.\n\nComments on the methods: Well written\nGeneral support for abortion provision section:\n\nThere is growing research that clinical exposure to abortion in medical school improves student education and interest in abortion. See: https://pubmed.ncbi.nlm.nih.gov/36375261/1\n\nIt will make your commentary stronger to comment on existing literature which your study either supports or goes against. It will make it more relevant to the current field of literature.\nMore comments:\nMaybe an additional conclusion can be urging that medical schools expose students to abortion clinically so that students as future physicians may make their own informed choices and women's choices will be better supported\n\nStrengths and limitations\nAgree not generalizable\n\nI would comment on your response rate of 25% being quite low. Most acceptable response rates for top journals are > 50% which means there may be significant response bias here. This needs to be mentioned.\n\nIs the work 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": "179602",
"date": "05 Jul 2023",
"name": "Mahmoud Alfaqih",
"expertise": [
"Reviewer Expertise My major area of interest is diet/lifestyle and how it affects the risk of chronic disease. However",
"I was involved in several studies that evaluated the viewpoints of the Jordanian population toward ethically convoluted topics related to reproduction such as surrogate pregnancy and abortion and COVID-19 vaccine acceptance."
],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address 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 discusses the attitude of medical students from one single institution of the Kingdom of Saudi Arabia toward induced abortion. The topic under study is a sensitive issue in the region and the literature that surrounds it is very scarce making the study very timely and of sufficient novelty to be considered for publication. The methods used are appropriate and the paper is generally well-structured. However, the manuscript has some major shortcomings. For example, more relevant and recent literature should be cited. Moreover, throughout the manuscript, there are several language and syntax errors. A professional round of language editing is recommended prior to its indexing.\nHere are some comments/suggestions to enhance the presentation of the manuscript.\nAbstract The abstract usually starts with some background information of the topic under study and the rationale/aims of why the investigation was conducted rather than just a definition of what abortion is.\nIntroduction\nDefinition of spontaneous abortion should be more precise and include the age of the fetus (before 20 weeks of gestational age).\n\nMany facts are presented, but very few references are provided. All information in the introduction needs to be cited.\n\nThe introduction needs to be expanded to include previous studies and published work on this topic. This study was performed in Saudi Arabia, are there any studies reporting the perception and attitudes of this population toward abortion? Are there any studies in the Middle East or Arab world? E.g. Alharbi et al., (2022)2 and Ibrahim et al., (2020)2. What were the findings of these studies?\nThe authors should discuss the laws and regulations related to abortion in Saudi Arabia and how they compare to other countries in the region and worldwide.\nMethods\nIt is more appropriate not to start your sentences with a number. Please modify accordingly: “1227 medical students were studying at the College of Medicine of the Imam Abdulrahman Bin Faisal University at the time when the study was conducted”.\n\nSample size calculation: please provide studies/numbers used for sample size calculation with appropriate citations. Was the response rate included in the calculation? What was the presumed response rate?\nResults\nThe text provides results stratified by gender, marital status, and academic level. However, only stratification by gender was provided in the tables.\n\nThere are some pieces of data presented in the text which are not provided in the tables.\n\nThere is inconsistency in reporting decimal places (some percentages are reported with one decimal place while others were reported as integers), there are multiple examples in all tables (table 1-4).\n\nIn the section “Conditional support for abortion provision”, it is reported that 248 (80.8%) participants were in favor of providing abortion in case of congenital anomalies not compatible with life. Calculating the percentage by dividing 248 by the no. of participants (308) results in a percentage of 80.5%.\n\nPlease revise all figures and percentages, other examples exist.\n\nThe text should only discuss the most important sections of the tables.\nDiscussion The discussion was a simple reiteration of the results of the study and was not insightful.\nConclusion There is no need to report percentages in the conclusions section.\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nPartly\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Partly",
"responses": []
}
] | 1
|
https://f1000research.com/articles/12-25
|
https://f1000research.com/articles/12-22/v1
|
09 Jan 23
|
{
"type": "Research Article",
"title": "Novel Helicobacter pylori-associated hemolysis Hp0499 and Hp1490 and its association with severity of gastritis",
"authors": [
"Sang Ketut Widiana",
"Titong Sugihartono",
"Dalla Doohan",
"Langgeng Agung Waskito",
"Kartika Afrida Fauzia",
"Yudith Annisa Ayu Rezkitha",
"Adi Wasis Prakosa",
"Ricky Indra Alfaray",
"Camilia Metadea Aji Safitri",
"Rizki Amalia",
"Yoshio Yamaoka",
"Muhammad Miftahussurur",
"Sang Ketut Widiana",
"Titong Sugihartono",
"Dalla Doohan",
"Langgeng Agung Waskito",
"Kartika Afrida Fauzia",
"Yudith Annisa Ayu Rezkitha",
"Adi Wasis Prakosa",
"Ricky Indra Alfaray",
"Camilia Metadea Aji Safitri",
"Rizki Amalia"
],
"abstract": "Background: Gastritis is an inflammation of the stomach lining often caused by Helicobacter pylori infection. Among three H. pylori genes coding for hemolytic toxins, the clinical outcome of hp0499 and hp1490 is unclear. We conducted molecular and histological analyses to evaluate the correlation between these genes and gastritis severity. Methods: We analyzed the hp0499 and hp1490 variants of 116 Indonesian samples using next generation sequencing and validated them using polymerase chain reaction (PCR). The updated Sydney system was used to grade gastritis through histological analyses. We then calculated the influence of hp0499 and hp1490 on the gastritis severity, using multivariate analysis and cagA and vacA as major H. pylori virulence factors. Results: Two variants of each gene were identified and named hp0499-1 and -2, and hp1490-1 and -2. We noted that hp0499 expression was significantly correlated with corporal atrophy (p = 0.037). H. pylori hp1490 significantly correlated with antral acute and chronic inflammation as well as corporal density (p = 0.025, p = 0.07, p = 0.010, respectively). After adjusting for age and sex, we found that vacA s1m1 was an independent risk factor for acute antral inflammation (p = 0.032). hp1490 and vacA s1m1 were independent risk factors for chronic antral inflammation (p = 0.030 and p = 0.031, respectively). Conclusions: We identified the variants hp0499-1 and -2 and hp1490-1 and -2 and demonstrated that hp0499 plays a significant role in the severity of corporal atrophy. Moreover, hp1490 was characterized as an independent risk factor for chronic inflammation in the antral region. Therefore, hp0499 and hp1490 are new potential targets for therapeutics.",
"keywords": [
"Helicobacter pylori",
"hp0499",
"hp1490",
"gastritis",
"infectious disease"
],
"content": "Introduction\n\nGastritis is an inflammation of the stomach lining. Several factors can cause this condition, including Helicobacter pylori infection, autoimmunity, nonsteroidal anti-inflammatory drugs, bile reflux, and allergic responses. In the histological observation, inflammatory cells are present in the stomach mucosa and submucosa.1 According to the World Health Organization (WHO), gastritis affects roughly 1.8–2.1 million people worldwide each year, or 0.03% of the global population.2 The infection prevalence is high in many areas such as Africa and East Asia, with rates of 79.1% and 56.1%.3 In a study from Indonesia, approximately 46% of all patients referred for upper endoscopy had gastritis.4 According to a recent survey, dyspepsia, one of the main symptoms of gastritis, was ranked sixth among the top 10 disorders that occurred in hospitalized Indonesian patients.5 However, there are limited healthcare facilities that provide gastrointestinal endoscopy services.\n\nH. pylori is the most frequent pathogenic cause of gastritis.6 This bacterium is strongly associated with the prevalence of gastric and duodenal ulcers, primary B-cell gastric lymphoma, gastric carcinoma, and mucosa-associated lymphoid tissue lymphoma.7 Furthermore, numerous adhesins, such as sialic acid-binding adhesin A, blood group antigen-binding adhesin A (BabA), and outer inflammatory protein A (OipA) have been found to be produced by H. pylori.8 CagA and VacA genes are groups of non-adhesin virulence factors that, alongside the adhesin groups, have a massive part in the H. pylori pathogenesis.9 In addition, low-virulence H. pylori can induce inflammation and maintain colonization ability. It is also characterized by the presence of vacA s2, agA-negative, off-type oipA, and absence of babA genes. This suggests the involvement of other virulence factors, which include the Hp0499 and Hp1490 toxins.10\n\nThe mechanism of action of Hp1490 is not known. In contrast, Hp0499 has been identified as a phospholipase A (PldA), which has enzymatic and hemolytic activity through phospholipase (PL) enzymes as the main factor, and is considered a degrading component of the mucosal barrier. Members of the PL enzymes, such as PLC, PLA1, and PLA2, are found in H. pylori. These enzymes are crucial for degrading the integrity of the gastric layers. PL enzymes hydrolyze membrane phospholipids at a specific concentration. This process induces an inflammatory response, causing gastritis.10 A mutation study in the pldA gene strongly suggested that H. pylori PLs are a critical determinant of virulence.11 Another study also stated that the high levels of PLA2 and lysolecithin in gastric juices from individuals with H. pylori infection support the hypothesis that PLA2 is involved in inflammation and mucosal damage associated with gastric ulcer formation.12\n\nThe prevalence of H. pylori infection in Indonesia is modest, ranging from 10.4% to 22.1%,13 and its virulence is low. This can be proven by the most common genotypes being the East Asian-type cagA with a 6-bp deletion in the pre-EPIYA region, as well as the jhp0562/-(1,3) gal T double-positive, dupA negative/short-type dupA, and vacA m2 and dupA negative/short-type dupA.14 Moreover, previous Indonesian studies have indicated a large variety in the prevalence and clinical manifestations of H. pylori infection between certain ethnic groups. The presence of additional virulence factors could be the cause of these disparities.15\n\nTherefore, this study aimed to analyze and find the correlation between the sequences of hp0499 and hp1490 in the Indonesian H. pylori strains. This study also aimed to investigate hp0499 and hp1490 as potential targets for developing new gastritis treatments.\n\n\nMethods\n\nWritten informed consent was obtained from all patients admitted to the study. All study protocols were approved by the Ethics Committees of the Medicine Faculty, Universitas Airlangga-Dr. Soetomo Teaching Hospital, Surabaya, Indonesia (221/Panke.KKE/IX/2012), Dr. Cipto Mangunkusumo Teaching Hospital, Jakarta, Indonesia (206/112/P1/ETIK/2014), Dr. Wahidin Sudirohusodo Teaching Hospital, Makassar, Indonesia (0208/H4.8.4.5.31/PP36-KOMETIK/2015), and the Oita University Faculty of Medicine, Yufu, Japan (P-12-10). The written informed consent was obtained from all subjects involved in the study.\n\nThis was an observational analytical study using a cross-sectional approach. Similar to our previous study,14 H. pylori was isolated from the tissues obtained via the corporal and antral gastric biopsies. We analyzed the hp0499 variants of 116 H. pylori DNA samples in total. We also analyzed 1172 hp1490 sequences from dyspepsia patients who underwent endoscopy in 19 different cities in Indonesia, ranging from August 2012 to March 2017, which have been studied by next-generation sequencing (NGS) and confirmed by conventional polymerase chain reaction (PCR, Figure S1, Extended data).9,16–18 These data were used to exclude non-fasted patients with partial or total gastrectomy and those contraindicated for upper endoscopy.\n\nWritten informed consent was obtained from all patients. All study protocols were approved by the Ethics Committees of the Faculty of Medicine, Universitas Airlangga, Dr. Soetomo Teaching Hospital (Surabaya, Indonesia), Dr. Wahidin Sudirohusodo Teaching Hospital (Makassar, Indonesia), Dr. Cipto Mangunkusumo Teaching Hospital (Jakarta, Indonesia), and the Oita University Faculty of Medicine (Yufu, Japan). All research procedures were based on the national and institutional ethical standards of human-based experimentation and the Helsinki Declaration of 1975, as revised in 2008 and 2013.\n\nDNA was obtained from H. pylori bacteria for the molecular analysis and used to sequence hp0499 and hp1490, and the virulence factors vacA and cagA. Takara Taq DNA Polymerase and Premix were used to amplify these genes (TaKaRa Taq version 2.0, Tokyo, Japan) with different primer sets including ND5F (5′ AGGTGGATTATAACTACTATTTGCGC 3′) and ND5R (5′ CCATCCCCATAGCCGTTAAAC 3′) primers for hp0499,11 and MD8F (5′ ATG GGGAGGAATCAAGGA 3′) and MD8R (5′ TCATGCTTCATTTTCTCCCT 3′) primers for hp1490. The PCR mixture contained 2 μL DNA (100 ng/μL), 0.125 μL Takara Taq DNA Polymerase (5 unit/μL), 1 μM of each primer (previously mentioned), 2.5 μL buffer, and 17.38 μL distilled water. The reaction was submitted to a pre-denaturation at 95 °C for 2 min, followed by 34 cycles at 95 °C for 30 s, 54 °C for 30 s, and 72 °C for 1 min, and a final extension step at 72 °C for 2 min. The resulting products were analyzed by electrophoresis using a 1.5% agarose gel containing ethidium bromide. It was visualized under ultraviolet light using the imager ChemiDoc XRS+ (Bio-Rad, Hercules, California, USA). Positive hp0499 and hp1490 strains were identified by the presence of amplicons of approximately 1068 bp and 1350 bp, respectively.\n\nWhole-genome data of 1172 hp1490 sequences that have been mentioned previously in methods and 116 new hp0499 sequences16 were used in this study to analyze the correlation between hp0499 and hp1490 sequences. Acceptable sequence data was obtained by trimming the raw FASTQ data using Trimmomatic software.19 Then, SPAdes (version 3.12)20 was used to combine the high-quality sequencing data. The BLAST tool was used for the hp0499 and hp1490 probable hemolysin gene sequences.21 After sequence alignment with other H. pylori reference strains, including the H. pylori 26695,22 the neighbor-joining method with 500 bootstrapping from the Molecular Evolutionary Genetic Analysis X program23 was used to obtain a phylogenetic tree. The homology between Hp0499 and Hp1490 amino acid sequences from these different H. pylori strains was analyzed using the Bioedit 7.2 software and the pairwise alignment method.24\n\nBased on the updated Sydney system guidelines,25 gastritis severity was assessed using the histopathological description of the gastric mucosa inflammation for neutrophil activity, intestinal metaplasia, atrophy, inflammation, and bacterial density. Two histopathological preparations were required for each sample and were observed under a microscope at 400 ×magnification. The microscopic characteristics of each sample were observed in five different view fields and used to classify the samples as grade 0, for no acute and chronic inflammatory cells, no tissue metaplasia, no glandular atrophy, and absence of H. pylori; and as grades I, II, and III for mild, moderate, and marked/severely damaged tissues and H. pylori presence, respectively. It was ensured that the research was carried out on at least one antrum sample and one corpus sample.\n\nBuffered formalin was used to fix all the biopsy samples. Paraffin was then used to embed samples for histological testing. Hematoxylin and eosin and May-Giemsa staining were used for specific sections. With that, we could assess the infiltration of monocytes or neutrophils to define the gastritis-driven mucosal damage, and the precancerous lesions to define intestinal metaplasia atrophic gastritis. These findings are further classified based on the updated Sydney system guidelines (Figure S2a-b, Extended data).25 We have also performed immunohistochemistry of all samples to increase the accuracy of H. pylori detection (Figure S2c). The specimens in this study were evaluated by an experienced pathologist (Tomohisa Uchida) who also performed similar studies in Vietnam, Myanmar, the Dominican Republic, Indonesia, and Bhutan.14\n\nThe IBM SPSS version 20.0 was used to analyze the data. The Chi-square test or Fisher’s exact test was performed to test the variables. Statistical significance was set at p < 0.05. The multivariate regression method was also performed using all determinants with p < 0.05. A multivariate logistic regression model calculated the odds ratios (OR) of clinical outcomes and H. pylori infection by age and sex. Lastly, the odds estimations were assessed using 95% confidence intervals (CI) and odds ratios.\n\n\nResults\n\nIn previous studies conducted at several endoscopy centers throughout Indonesia, 116 H. pylori specimens with severity diagnoses were obtained. We also acquired an additional 104 samples of hp0499 H. pylori strains and 106 samples of hp1490 H. pylori strains, with which we performed a homology test with a pairwise alignment, as sequencing limitations for hp0499 and hp1490 were common. The evolutionary correlation between the hp0499 and hp1490 sequences was constructed through phylogenetic analysis. The constructed tree showed the two main branches of hp0499 and hp1490 (Figure 1). The first upper branch of hp0499 consisted of 79 samples and one reference strain, H. pylori 26695 (Figure 1A). The hp1490 gene was clustered in two main branches (Figure 1B). The first branch comprised 73 samples, and the second contained 35 samples and the reference strain H. pylori 26695. In addition, as a reference to another study naming the two groups or variants of hp1490 was not found, the first variant was named variant hp1490-1, while the second was named variant hp1490-2.\n\n(A) hp0499 phylogenetic tree. (B) hp1490 phylogenetic tree.\n\nThe relationship analysis between the hp0499 and hp1490 H. pylori types and the gastric mucosal grade as well as the density of bacteria was performed (Figures 2-5). All histopathological parameters were assessed in the antrum and corpus.\n\nIM: Intestinal metaplasia.\n\nIM: Intestinal metaplasia.\n\nIM: Intestinal metaplasia.\n\nIM: Intestinal metaplasia.\n\nFor hp0499, we found that only the atrophy parameter in the corpus was significantly associated with the hp0499 variant (p = 0.037), whereas other histopathological parameters did not show a significant correlation (Figures 2 and 3).\n\nWe analyzed hp1490 using the updated Sydney system, and the hp1490 variant was found to have a significant association with the parameters of acute and chronic antrum inflammation along with the density of H. pylori in the corpus region (p = 0.025, p = 0.07, p = 0.010, respectively). In contrast, other histopathological parameters did not show a significant correlation (Figures 4 and 5).\n\nSince we observed a significant relationship between the variance of hp0499 and hp1490 and gastric mucosal status, we performed a multivariate analysis (Table 1) including the main virulence factors of H. pylori cagA and vacA.26 In our study, we found that vacA was significantly associated with antral acute and chronic inflammation (p < 0.05). We then performed multivariate analysis by including vacA and the hp1490 variant, adjusting for age and sex. We found that vacA s1m1 was an independent risk factor for acute antral inflammation (p = 0.032), as well as for chronic antral inflammation (p = 0.031).\n\n\nDiscussion\n\nOnly a few researchers have examined the relationship of H. pylori hp0499 and hp1490 with the pathophysiology of H. pylori infectivity and consequent gastritis severity, as the major limitation is the availability of molecular and structural characterization of these genes. So far, there has been no population-level research on hp0499 or hp1490. In addition, previous studies could only identify particular strains that carry these genes.16 Our study showed that H. pylori isolates from Indonesia contain the hp0499 and hp1490 genes. These findings indicate that the majority of H. pylori strains contain hp0499 and hp1490, suggesting that every H. pylori strain may cause hemolysis, as hp0499 and hp1490 encode hemolytic-related toxins.21 Previous studies have shown that hemolytic activity is critical for the survival of various bacteria. This includes Listeriolysin O, a type of hemolysin produced by Listeria monocytogenes. This hemolysin type also induces the process of bacterial release from phagosomes into the cytosol. It promotes the secretion of HlyA, a hemolysin produced by Escherichia coli which has been proven to elevate pro-inflammatory cytokine levels, such as IL-6 and IL-8, during colonization and infection processess.27 However, only a limited number of studies have explained the mechanism of hemolysis caused by H. pylori and the role of the components involved in this process.\n\nThe phylogenetic analysis showed that hp0499 and hp1490 are classified into two major monophyletic groups. Due to the lack of literature on the hp0499 and hp1490 variants of H. pylori, we hypothesized that the first monophyletic group on hp0499 is a hp0499-1 variant and the second is hp0499-2, while hp1490 clades are respectively the hp1490-1 and hp1490-2 variant groups. Previously, some genotypes of H. pylori virulence genes, such as cagA, were associated with geographical regions.28 Varieties of predominant cagA types were observed in different regions, with the East Asian type being the most predominant one in Indonesia.14,16 Our data also showed similar results, with 57% of the H. pylori cagA type being East Asian samples. The remaining samples were ABB-type (16%) and Western-type (27%). The vacA profile was dominated by the s1m1 genotype, which accounted for 68% of the samples. Further studies are necessary to structurally characterize the proteins of these genes.\n\nOur study found a significant association between hp0499 and parameters of corporal atrophy and acute inflammation, chronic antrum inflammation, and density of H. pylori in the corpus, which was significantly correlated with the hp1490 type. The exact mechanisms of these two hemolytic-associated virulence factors are still not clearly understood. Another hemolysin factor, tylA, showed a significant association with the density of the bacteria, suggesting a more prominent role in colonization rather than inflammation.29 In addition, a previous report showed that both pldA- and tlyA-negative mutants could efficiently colonize a mouse model of H. pylori. This finding suggests that hemolysin activity is significantly associated with the colonization process and subsequent pathogenic pathway activation.27 H. pylori is a unique bacterium that harbors many virulence factors in its genome. Among these virulence factors, the most widely studied are vacA and cagA. These two virulence factors are considered the golden standard or main factors needed to determine the diagnosis.30,31 In addition to these, several other virulence factors are known to play an essential part in the H. pylori pathogenesis, such as dupĀ, oipA, babA, and iceA, each one with a different role and mechanism of action.30,31\n\nThis study performed multivariate analysis between the gold standard H. pylori virulence factors cagA and vacA and acute and chronic inflammation parameters in the antrum against hp1490. The bivariate analysis showed that cagA and vacA have a significant association with acute and chronic inflammation parameters in the antrum against hp1490. This raises the suspicion that the pathophysiology of cagA and vacA is related to these bacterial types’ colonization and hemolytic processes in the stomach, as well as hp1490 type. This also demonstrates the interaction between hp1490 with cagA and vacA in the overall pathogenesis of H. pylori infection.\n\nDespite the present study's limitation due to its small sample size, our study provides the latest profile information about hp0499 and hp1490 and their role in the H. pylori pathogenesis. Additionally, all samples were from Indonesia, which has a relatively small prevalence of gastric cancer and gastritis atrophy. Unfortunately, our results cannot be generalized to populations in other countries due to this limitation. Hence, it is necessary to analyze additional samples and broaden the current investigation to other countries with a higher prevalence of gastric cancer and gastritis. Furthermore, research on the structure, binding, and attached proteins of hp0499 and hp1490 is crucial, as well as their association with other virulence factors, also need to be studied in the near future.\n\n\nConclusions\n\nWe observed two hp0499 and hp1490 variants (hp0499-1 and -2, and hp1490-1 and -2) in the H. pylori strains from Indonesia. A significant association of hp0499 with atrophy in the corpus suggested that hp0499 plays a larger role in inflammation in the gastric mucosa. Meanwhile, hp1490 was significantly associated with acute inflammation, chronic inflammation of the antrum, and density of H. pylori bacteria in the corpus, suggesting that hp1490 contributes more to colonization rather than inflammation in the gastric mucosa.",
"appendix": "Data availability\n\nNCBI GenBank: Helicobacter pylori strain IND07_01290 phospholipase A1 (HP0499) gene, complete cds. Accession number: OP756069 (BioProject: PRJEB53580); https://identifiers.org/insdc:OP756069. 32\n\nNCBI GenBank: Helicobacter pylori strain TER9_01326 phospholipase A1 (HP0499) gene, partial cds. Accession number: OP756181 (BioProject: PRJEB53580), https://identifiers.org/insdc:OP756181. 33\n\nNCBI GenBank: Helicobacter pylori strain IND07_00061 hypothetical protein (HP1490) gene, complete cds. Accesion number: OP756182 (BioProject: PRJEB53580); https://identifiers.org/insdc:OP756182. 34\n\nNCBI GenBank: Helicobacter pylori strain TER9_00758 hypothetical protein (HP1490) gene, complete cds. Accession: OP756291 (BioProject: PRJEB53580); https://identifiers.org/insdc:OP756291. 35\n\nFigshare: Dataset Novel Helicobater pylori and its association with severity of gastritis, https://doi.org/10.6084/m9.figshare.20059079. 36\n\nThis project contains the following underlying data:\n\n- Dataset Novel Helicobacter pylori associated hemolysis Hp0499 and Hp1490.xls\n\nFigshare: Dataset Novel Helicobater pylori and its association with severity of gastritis, https://doi.org/10.6084/m9.figshare.20059079. 36\n\nThis project contains the following extended data:\n\n- hp0499 dan hp1490-jpeg. (Phylogenetic trees)\n\n- Figure S1a_Electrophoresis image hp0499.tif\n\n- Figure S1b_Electrophoresis image hp1490.tif\n\n- Figure S2a_Histological Test Image.JPG\n\n- Figure S2b_Histological Test Image.JPG\n\n- Figure S2c_Immunohistochemistry results. JPG\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 the Faculty of Medicine, Universitas Airlangga, and the Dr. Soetomo Teaching Hospital, Surabaya, Indonesia.\n\n\nReferences\n\nSetiawati S, et al.: Buku Ajar Ilmu Penyakit Dalam Jilid I Edisi VI. Ideas Publishing;2019.\n\nGustin RK: Faktor-faktor Yang Berhubungan dengan Kejadian Gastritis pada Pasien yang Berobat Jalan di Puskesmas Gulai Bancah Kota Bukittinggi Tahun 2011. Artikel Penelitian;2011; 1–12.\n\nHooi JKY, Lai WY, Ng WK, et al.: Global prevalence of Helicobacter pylori infection: systematic review and meta-analysis. Gastroenterology. 2011.\n\nMiftahussurur M, et al.: Analysis of risks of gastric cancer by gastric mucosa among Indonesian ethnic groups. PLoS One. 2019; 14(5): e0216670–e0216619. PubMed Abstract | Publisher Full Text | Free Full Text\n\nAbdullah M, et al.: Prevalence, risk factors and socio-epidemiological study of gastroesophageal reflux disease: An Urban population based study in Indonesia. Asian J. Epidemiol. 2016; 9(1–3): 18–23. Publisher Full Text\n\nUchida T, et al.: Helicobacter pylori infection in Thailand: A nationwide study of the CagA phenotype. PLoS One. 2015; 10(9): e0136775. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMilani M, et al.: The status of antimicrobial resistance of Helicobacter pylori in Eastern Azerbaijan, Iran: comparative study according to demographics. J. Infect. Chemother. 2012; 18(6): 848–852. PubMed Abstract | Publisher Full Text\n\nOleastro M, Ménard A: The role of Helicobacter pylori outer membrane proteins in adherence and pathogenesis. Biology. 2013; 2(3): 1110–1134. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMiftahussurur M, Yamaoka Y, Graham DY: Helicobacter pylori as an oncogenic pathogen, revisited. Expert Rev. Mol. Med. 2017; 19: e4. PubMed Abstract | Publisher Full Text | Free Full Text\n\nLata K, Paul K, Chattopadhyay K: Functional characterization of Helicobacter pylori TlyA: Pore-forming hemolytic activity and cytotoxic property of the protein. Biochem. Biophys. Res. Commun. 2014; 444(2): 153–157. PubMed Abstract | Publisher Full Text\n\nDorrell N, et al.: Characterization of Helicobacter pylori PldA, a phospholipase with a role in colonization of the gastric mucosa. Gastroenterology. 1999; 117: 1098–1104. PubMed Abstract | Publisher Full Text\n\nLangton SR, Cesareo SD: Helicobacter pylori associated phospholipase A2 activity: A factor in peptic ulcer production? J. Clin. Pathol. 1992; 45(3): 221–224. PubMed Abstract | Publisher Full Text | Free Full Text\n\nSyam AF, et al.: Risk factors and prevalence of Helicobacter pylori in five largest islands of Indonesia: A preliminary study. PLoS One. 2015; 10(11): e0140186. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMiftahussurur M, et al.: Helicobacter pylori virulence genes in the five largest islands of Indonesia. Gut Pathog. 2015; 7: 26. 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. Nature Publishing Group. PubMed Abstract | Publisher Full Text | Free Full Text\n\nWaskito LA, et al.: Distribution and clinical associations of integrating conjugative elements and cag pathogenicity islands of Helicobacter pylori in Indonesia. Sci. Rep. 2018; 8(1): 6073–6079. PubMed Abstract | Publisher Full Text | Free Full Text\n\nDoohan D, et al.: Characterization of a novel Helicobacter pylori East Asian-type CagA ELISA for detecting patients infected with various cagA genotypes. Med. Microbiol. Immunol. 2020; 209(1): 29–40. Springer Berlin Heidelberg. PubMed Abstract | Publisher Full Text | Free Full Text\n\nFauzia KA, et al.: Biofilm formation and antibiotic resistance phenotype of Helicobacter pylori clinical isolates. Toxins. 2020; 12(8). PubMed Abstract | Publisher Full Text | Free Full Text\n\nBolger AM, Lohse M, Usadel B: Trimmomatic: A flexible trimmer for Illumina sequence data. Bioinformatics. 2014; 30(15): 2114–2120. PubMed Abstract | Publisher Full Text | Free Full Text\n\nBankevich A, et al.: SPAdes: A new genome assembly algorithm and its applications to single-cell sequencing. J. Comput. Biol. 2012; 19(5): 455–477. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMartino MC, et al.: Helicobacter pylori pore-forming cytolysin orthologue TlyA possesses in vitro hemolytic activity and has a role in colonization of the gastric mucosa. Infect. Immun. 2001; 69(3): 1697–1703. PubMed Abstract | Publisher Full Text | Free Full Text\n\nTomb JF, et al.: Erratum: The complete genome sequence of the gastric pathogen Helicobacter pylori (Nature (1997) 388 (539-547)). Nature. 1997; 389(6649): 412. Publisher Full Text\n\nKumar S, et al.: MEGA X: Molecular evolutionary genetics analysis across computing platforms. Mol. Biol. Evol. 2018; 35(6): 1547–1549. PubMed Abstract | Publisher Full Text | Free Full Text\n\nHall T, Biosciences I, Carlsbad C: BioEdit: An important software for molecular biology. GERF Bull. Biosci. 2011; 2: 60–61.\n\nAydin O, et al.: Interobserver variation in histopathological assessment of Helicobacter pylori gastritis. World J. Gastroenterol. 2003; 9(10): 2232–2235. PubMed Abstract | Publisher Full Text | Free Full Text\n\nCastillo-Rojas G, Mazarí-Hiriart M, López-Vidal Y: Helicobacter pylori: focus on CagA and VacA major virulence factors. Salud Publica Mex. 2004; 46(6): 538–548. PubMed Abstract | Publisher Full Text\n\nJavadi MB, Katzenmeier G: The Forgotten Virulence Factor: The “non-conventional” Hemolysin TlyA And Its Role in Helicobacter pylori Infection. Curr. Microbiol. 2016; 73(6): 930–937. PubMed Abstract | Publisher Full Text\n\nYamaoka Y: Helicobacter pylori typing as a tool for tracking human migration. Clin. Microbiol. Infect. 2009; 15(9): 829–834. PubMed Abstract | Publisher Full Text | Free Full Text\n\nPrakosa AW, et al.: Characterization of Helicobacter pylori tlyA and Its Association with Bacterial Density. Dig. Dis. 2021; 40: 417–426. PubMed Abstract | Publisher Full Text\n\nHirlan: Buku Ajar Ilmu Penyakit Dalam, Jilid I, edisi V. Jakarta:Pusat Penerbitan Departemen Ilmu Penyakit Dalam FK-UI;2009.\n\nKim JM: H. pylori Virulence Factors: Toxins (CagA, VacA, DupA, OipA, IceA). Biol. Med. 2016.\n\nWidiana, et al.:Helicobacter pylori strain IND07_01290 phospholipase A1 (HP0499) gene, complete cds. [data set].2022. NCBI GenBank, Accession number: OP756069.Reference Source\n\nWidiana, et al.:Helicobacter pylori strain TER9_01326 phospholipase A1 (HP0499) gene, partial cds. [data set].2022. NCBI GenBank, Accession number: OP756181.Reference Source\n\nWidiana, et al.:Helicobacter pylori strain IND07_00061 hypothetical protein (HP1490) gene, complete cds. [data set].2022. NCBI GenBank, Accession number: OP756182.Reference Source\n\nWidiana, et al.:Helicobacter pylori strain TER9_00758 hypothetical protein (HP1490) gene, complete cds. [data set].2022. NCBI GenBank, Accession number: OP756291.Reference Source\n\nWidiana SK, Sugihartono T, Doohan D, et al.:Dataset Novel Helicobater pylori and its association with severity of gastritis. [Data set]. figshare. 2022. Publisher Full Text"
}
|
[
{
"id": "165206",
"date": "30 Mar 2023",
"name": "I Ketut Mariadi",
"expertise": [
"Reviewer Expertise Gastroenterology"
],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 excellent and gives an important addition to the literature. This interesting research reports new virulence factors, including the Hp0499 and Hp1490 toxins. Some improvements should be made:\nSome literature was published older than five years, and better to cite from the original publication (ex: ref no 2 in the introduction).\n\nThe size effect of the analysis should be mentioned clearly, not just presenting the p-value.\n\nThe size effect (OR in multivariate analysis) should be given an appropriate interpretation, not just mentioning there is a correlation.\n\nIn conclusion, no clear data support the conclusion. ex: \"A significant association of hp0499 with atrophy in the corpus suggested that hp0499 plays a larger role in inflammation in the gastric mucosa\"\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": []
}
] | 1
|
https://f1000research.com/articles/12-22
|
https://f1000research.com/articles/12-18/v1
|
06 Jan 23
|
{
"type": "Systematic Review",
"title": "Pharmacy students' empathy and its determinants: a systematic review",
"authors": [
"Hening Pratiwi",
"Susi Ari Kristina",
"Anna Wahyuni Widayanti",
"Yayi Suryo Prabandari",
"Hening Pratiwi",
"Anna Wahyuni Widayanti",
"Yayi Suryo Prabandari"
],
"abstract": "Background: Empathy in the context of healthcare is an immersion experience to comprehend patients' viewpoints, feelings, and emotions, without passing judgment, to ensure they receive the necessary treatment to feel comfortable. Empathy for others must be possessed by healthcare professionals and healthcare students as healthcare professionals’ candidates, including the pharmacy student. This study aimed to identify and assess the determinants related to pharmacy students' empathy. Methods: Three electronic databases were used for the first searches. We used peer-reviewed original papers, full text, must assess determinants that are associated with pharmacy students' empathy, and only be focused on pharmacy students (first to the fourth year) as healthcare professionals candidates. We utilized Joanna Briggs Institute Critical Appraisal Checklists to observe the quality of published publications and reduce bias. Results: This review examined 14 papers that reported on determinants connected to pharmacy students' empathy. Nine studies evaluated the association between sex and the level of empathy, seven studies reported educational intervention, four studies discussed the year of study, two studies explained the type of school, four studies evaluated experience, and others determinants that discussed in the included studies were career preference, intercultural sensitivity, stigma, altruism, grit, self-awareness, marital status, and family income Conclusions: Educational intervention, experience, gender or sex, type of school, year of study, intercultural sensitivity, career preference, altruism, grit, self-awareness, marital status, and family income, can all have a positive impact on increased empathy among pharmacy students. We acknowledge that the included studies are heterogeneous, indicating that additional studies are necessary before reaching any firm conclusions. More research is needed to properly understand how empathy can be improved with the most effective pharmacy educational strategies. Higher levels of evidence are also required in studies to address the potential bias caused using self-report questionnaires, as well as other potential biases and inaccuracies.",
"keywords": [
"empathy",
"pharmacy students",
"factors",
"determinants",
"educational intervention",
"experience"
],
"content": "Background\n\nEmpathy, in the context of healthcare, is an emotional response experienced when attempting to comprehend patients' viewpoints, feelings, and emotions without passing judgment, to ensure they receive the necessary treatment to feel comfortable.1–3 These statements demonstrate three skills of healthcare professionals: expressing empathy, cognitive ability to recognize and comprehend the views and emotions of a patient, and behavioral ability to convey this understanding.4 Higher levels of empathy among healthcare professionals not only foster higher quality communication with patients, but they also result in favorable patient outcomes such as better patient self-care, higher patient satisfaction and faster recovery times.5 Indeed, having empathy is crucial for building closer relationships with patients and for understanding their needs, as is frequently stated in health and educational programs for healthcare professionals.6\n\nEmpathy for others must be possessed by healthcare professionals and healthcare students, as healthcare professional candidates in training, but several studies have explained that there can be a decrease in student empathy over the course of their medical training.7–9 Studies indicate that empathy declines as the student progresses through medical school,7,8 dentistry school,9,10 and pharmacy school,9,11 despite the significance of increasing empathy in health professions students. As personal discomfort from burnout, sadness, and diminished quality of life develops among trainees during their training, they are less likely to feel or show empathy. Neumann et al. found that deficits in the formal (e.g., lack of formal empathy training), informal (inadequate mentors and improper learning environments), and factors outside of the medical curriculum (e.g., abuse of students and high workload) medical curriculum may contribute to this decrease in empathy levels over time.7 These results serve as a warning to the faculty members and educators of educational institutions that strategies must be developed not only to stop the depletion of empathy but also to improve students' empathetic orientation to better comprehend patients.12 To develop a strategy, one must define the target needs and understand the factors or determinants that are related to student empathy, in this case being the pharmacy student. This review is designed to identify those of the pharmacy student. The growth of the pharmacy educational system was anticipated to broaden pharmacists' roles in patient-centered care.2,13 Because empathy is important for patient care, educational strategies should encourage the development of empathy during training.\n\nA previous systematic review was conducted by Maximiano-Barreto et al. to investigate the variables linked to empathy levels among health-related students and professionals.6 However, in the included studies there was not much evidence to indicate pharmacy students' empathy levels specifically. Quince et al. conducted a review in a similar context to determine the variables that could influence how empathy develops in medical students,14 although the scope of this review was limited by its predominant emphasis on medical students, whereas in this review the emphasis is placed more on the empathy of pharmacy students. The authors were interested in conducting a comprehensive review of determinants related to empathy with a focus on pharmacy students to fill this gap in the literature.\n\nThis study aimed to identify and assess the determinants related specifically to pharmacy students' empathy. This review sought to answer the following two questions:\n\n1) How is empathy measured?\n\n2) What determinants may relate to the development of pharmacy students’ empathy?\n\n\nMethods\n\nIn the early planning stage, the four authors (HP, SAK, AWW, and YSP) discussed the existing theory regarding the determinants related to students' empathy. The author reads several articles that discuss factors that may be related to student empathy. Previous studies identified “perspective-taking,” “compassionate care,” and “standing in patient's shoes” programs that provide healthcare students with information about the relevance of empathy and its role in patients treatment.15,16 Organizational culture, personal and interpersonal relationships, and demographic characteristics are all highly linked to increased empathy. The authors were interested in conducting a comprehensive review of determinants related to empathy with a focus on pharmacy students.16 This review followed the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines.17\n\nWe used the following inclusion criteria to evaluate each of the articles under consideration to respond to our research questions:\n\n1) They must be peer-reviewed original papers.\n\n2) They must be published in full text.\n\n3) They must only be focused on pharmacy students (first to the fourth year) as healthcare professionals candidates.\n\n4) They must assess determinants that are associated with pharmacy students' empathy.\n\n5) The subdivisions of empathy must include attitude, behavior, and perception.\n\n6) They must conduct design research, such as cross-sectional studies, cohort studies, case-control, randomized control trials, or quasi-experimental studies.\n\nExcluded from the systematic review were studies on animals, review articles, qualitative studies, commentary articles, letters to the editor, conference abstracts, books, guidelines, and theses.\n\nThe initial searches were conducted using three electronic databases: PubMed, Science Direct, and Scopus. From July 14, 2022, to the latest recent search on July 16, 2022, we considered articles on factors or determinants about pharmacy students' empathy from this three-database collection. There were no restrictions on publication ages throughout this period. Each database used the terms “factors” AND “pharmacy students” AND “empathy” as a single search term or in combination using Medical Subjects Heading (MeSH) terms with the Boolean operators. We also searched for additional reference materials by consulting the cross-references listed in the included publications via Google Scholar on August 16, 2022.\n\nThe retrieved studies from databases were screened for their title and abstracts by three authors (HP, SAK, and AWW) to ensure they fit the eligibility criteria. We used a common data extraction spreadsheet (Microsoft Excel) to chart the necessary data. To eliminate duplication, Mendeley Reference Manager was used to obtain every article. Next, full texts of selected studies were reviewed by three authors (HP, SAK, YSP) to determine their relevance. Discrepancies whether the title, abstract, and entire manuscript meet the inclusion and exclusion criteria were resolved through discussions between authors until concordance was obtained. A PRISMA diagram was used to record the screening and selection process (Figure 1).\n\nA data extraction was conducted by the two independent reviewers (HP and SAK) to obtain the key study information. Included studies should provide a primary focus on identifying the determinants which related to pharmacy student empathy. While other supporting variables include characteristics of included studies (the year of publication, country, study design, participants, and type of participants) and measurements adjusted for eligibility criteria.\n\nWe created a spreadsheet with details about the publication year, country, study design, participants, participant types, measurements, and factors related to empathy. Two authors (HP and SAK) read the entire paper and highlight the key points discussed inside it, with adjustments for the relevant variables. Furthermore, this pertinent information was reviewed in the results section. The two reviewers' disagreement (HP and SAK) were settled through discussion with another member of the research team (HP, SAK, AWW, YSP) and HP as the person in charge of this discussion process.\n\nOne point of disagreement is the assumption that the “country” is related to the country in which the study was conducted rather than the country of the affiliated university. Another example is figuring out the essential factors that related pharmacy students' empathy as indicated in the included studies.\n\nWe utilized Joanna Briggs Institute Critical Appraisal Checklists (JBI) according to the methodological design of the studies: cross-sectional studies, cohort studies, case-control, randomized control trials, and quasi-experimental studies. This is performed to observe the quality of published articles. The JBI critical appraisal method was used to determine the extent to which the included studies attempted to reduce risk of biases related to the appropriateness of the study objectives participant selection, data collection, data analysis, randomization, treatment allocation, blinding, and interpretation of results. There were 8 questions for a cross-sectional study design, 11 questions for a cohort study, 10 questions for a case-control study, 9 questions for a quasi-experimental study, and 13 questions for a randomized controlled trial.\n\nAppraisal of studies was undertaken by two independent authors (HP and SAK) and assessments were compared. The discrepancies were discussed with other research team members (AWW and YSP). Examples of discrepancies include checking to see if the inclusion criteria were explained clearly and checking to see which confounding variables were mentioned in the article.\n\nEach checklist criterion was rated as yes”, “no”, “unclear”, or “not applicable”. When the studies reached up to 49% of the “yes” score, the risk of bias was classified as high; moderate when they reached 50 to 69% of the “yes” score; and low when they reached more than 70% of the “yes” score (Joanna Briggs Institute Critical Appraisal Checklists).\n\n\nResults\n\nInitially, a total of 153 records were retrieved from databases and other methods (PubMed with 16 articles, Science Direct with 112, Scopus with 25, and citation searching from Google Scholar with 5). Titles and abstracts were evaluated after duplicate articles (137 articles from databases) were removed to see how they related to this systematic review. The study titles and abstracts were reviewed according to inclusion criteria i.e., they must be focused on pharmacy students (first to the fourth year) as healthcare professionals’ candidates, and they must assess determinants that are associated with pharmacy students' empathy. In cases where there was uncertainty regarding whether the title and abstract adequately express the determinants related to pharmacy students' empathy, the full text was read.\n\nDisagreements among the reviewers (HP, SAK, and AWW) on whether the title and abstract met the inclusion requirements were settled through discussion until consensus was reached. We held discussions by getting together and going over titles and abstracts that would meet our inclusion criteria. We read and discuss the entire article together, considering the key points for entering the included studies until we get to an agreement if an article is identified that is doubtful considering the inclusion criteria. Out of these, 120 items were excluded at this stage because they failed to fulfill the requirements for inclusion, including review articles, publications with populations of non-pharmacy students, and articles that were not pertinent to our topic. For example, several studies focus more on nursing students,18–20 another study focuses more on dietetic students.21\n\nA total of 22 articles were given a complete text examination and eligibility evaluation i.e. they must be peer-reviewed original papers, they must be published in full text, they must be focused on pharmacy students (first to the fourth year) as healthcare professionals’ candidates, and they must assess determinants that are associated with pharmacy students' empathy. The research team members (HP, SAK and YSP) independently reviewed the full texts of articles that fulfilled inclusion criteria and passed the title and abstract evaluation. The source population, participants type, sample size, study design, outcome measurement type, and determinants related to empathy were just a few of the metrics that were evaluated for each article. The research team's discussion led to the extraction and finalization of the data. We read and discussed the full text's information, particularly whether it included information on factors that influence empathy as well as supplementary details like the included studies' characteristics (year of publication, country, study design, participants, and type of participants) and measurements. Finally, 14 articles were included in this systematic review. Figure 1 provides an overview of the selection process.\n\nEach study that was considered, including an RCT, a quasi-experiment, and cross-sectional studies, had its quality and risk of bias evaluated critically. All studies obtained more than 50% “yes” responses on the checklist, and the risk of bias was graded as low, according to the author's quality evaluation using the JBI Quality appraisal checklist. The results of the quality assessment are shown in supporting information in data availability section.22\n\nThe included studies were published between 2011 and 2022 and were conducted in seven countries: eight in the United States2,23–29 and one each from Korea,30 China,31 United Kingdom,32 Indonesia,33 Singapore,34 and Iran.35 Eleven of the included studies used cross-sectional designs, two studies used randomized control trial designs,24,34 and one of the studies used one group pretest-posttest intervention design.28\n\nTwelve (86%) studies included pharmacy students as participants and two (14%) studies informed pharmacy students and other health professional students or pharmacists.28,33 Pharmacy students as participants ranged from first year through to fourth year students. Three of the included studies reported first-year level of pharmacy students as participants.2,26,29 Three studies reported second-year pharmacy students as participants.23,24,34 Two studies informed first through to fourth year students as participants.31,32 We found one paper focussed each on the third year,25 the fourth year,33 the second through to third year,30 the first through to third year,27 and two others that did not specify the education level of students were included in the study.28,35 The number of participants included in each of the studies ranged from 40 to 1013 pharmacy students.\n\nAll studies evaluated participant improvements in empathy using self-report measures. Self-report measurements typically consisted of a single question or a self-report survey. There were several different self-report survey formats used in included studies. The most frequently used instrument was the Jefferson Scale of Empathy for Health Professions Students (JSE-HPS).2,24,28–32,34,35 The JSE-HPS instrument contained 20 items with response options based on a 7-point Likert scale and measuring Perspective Taking, Compassionate Care, and Standing in the Patient's Shoes. Another instrument widely used in included studies was the Kiersma-Chen Empathy Scale (KCES).26,28,33 The KCES, a 15-item survey with a 7-point Likert-type scale, measured two aspects of empathy: the capacity to comprehend others' perspectives (cognitive domain) and the capacity to empathize with their thoughts and experiences (affective domain). Table 1 provides specifics about the empathy measurement tools used in included studies.\n\nGender or sex\n\nAmong the included studies in this review, eight studies evaluated the association between gender and the level of empathy2,27,29–32,34,35 and one study evaluated the association between sex and the level of empathy.26 Most of them concluded that the overall empathy score in female students was significantly higher than that in males,2,26,29,32,34,35 with two included studies discovering no discernible differences between them.30,31\n\nIn 2022, Fashami et al. conducted a cross-sectional study to measure the empathy score among 504 pharmacy students in five Iranian pharmacy schools.35 According to this study, female students scored much higher on empathy than males did overall. Additionally, in the JSE-HPS, females had considerably greater empathy scores in the compassionate care and perspective-taking domains. At the Midwestern University Chicago College of Pharmacy in 2011, Fjortoft et al. performed a survey of 187 first-year pharmacy students.2 According to this study, female pharmacy students scored much better on average for empathy than their male colleagues did. Jeon and Cho’s findings contrasted this by stating in their research that there were no noteworthy gender differences.30\n\nEducational intervention\n\nSeven included studies reported that educational intervention can become one of the factors or determinants related to the empathy of pharmacy students.23–25,28,29,31,34 Pharmacy students can develop their empathy using a variety of educational interventions, including but not limited to a learning module, role-reversal exercises, patient simulations, workshops, dramatic performances, and games involving patients’ medication.36 Lor et al. conducted a randomized study in which participants in the intervention group underwent a three-day simulation of a specific activity and received daily debriefings from faculty members.24 The three exercises involved were: simulating losing one's dominant hand (participants had to wrap their dominant hand in gauze and were not permitted to use it); simulating losing one's sight (participants had to wear sleep masks); and simulating losing one's ability to speak (participants were only allowed communicate using a whiteboard and marker). Seven days after the intervention, participants who were randomly assigned to a single, three-day empathy intervention experienced a significant rise in empathy scores.\n\nSimko et al. used the apparatuses contained in a Chronic Pain Simulation Empathy Training Kit (CPSETKit) to simulate chronic pain from several disease states.28 The purpose of this study was to assess how using a Chronic Pain Simulation Empathy Training Kit affected the empathy of pharmacy, nursing, and health sciences students (CPSETKit). The use of the CPSETKit increased the empathy for chronic pain patients in health care students. Additionally, Reed et al. investigated whether first-year pharmacy students' empathy levels enhanced after taking a longitudinal professionalism course at two pharmacy schools. The outcomes demonstrate that continuous professionalism training at two pharmaceutical schools raised the level of pharmacy students' empathy.29\n\nYear of study and type of school\n\nOf the 14 included studies, there were four studies30–32,35 discussed the year of study that related to the empathy of students, and two studies30,35 explained that the type of school related to the students’ empathy.\n\nLi et al. reported that in comparison to any prior years, the fourth-year students' mean empathy score was significantly higher than in other years but did not differ much.31 The second-year students also scored the worst on empathy. The fourth-year class had higher empathy scores than the first-year class, but there was no statistically significant difference in empathy scores by age, which may be because the seniors have finished all of their professional coursework and clinical practice. Due to the fact that pharmacy students in China spend their first three years away from the clinical setting, they seldom come into contact with clinical role models until their last year of training. Students work with patients during their fourth-year clinical clerkships while receiving training in ethics, practice management, and the management and treatment of fearful patients. As a result, they may come at this stage to deeply and vividly understand the significance of the relationship between pharmacists and patients.\n\nOn the other hand, Jeon & Cho reported that even though there were no differences across the students’ years of study in their research, the type of university attended determined significant differences in the total empathy score.30 Depending on whether the students came from co-educational or women's universities, significant variations were found for all variables. Students in women's universities exhibited higher levels of empathy overall, compassionate care, and perspective-taking than those at co-educational universities. Since 2011, the four-year BSc pharmacy degree in Korea has been replaced by a two+four hour Doctor of Pharmacy (PharmD) program. The first graduates of the new program will receive their diplomas in 2015. Students could take the Pharmacy Education Eligibility Test (PEET) if they have completed the minimum number of pre-pharmacy courses (two years).\n\nExperience\n\nA total of four studies of included studies evaluated the association between experience and the level of empathy.27,29,32,33 According to Williams et al., there were no appreciable differences in the cognitive empathy of student groups with direct patient care or chronic illness caregiving experiences.27\n\nSimilar to Hall et al., part-time employment experience affect students' empathy scores, even though mean scores were higher for part-time employees compared to non-employees.32 Additionally, students who said they worked with patients had a slightly higher mean empathy score than students who had not worked with patients. These findings are significant because they imply that students who have more patient exposure are not adversely affected by patient contact.\n\nReed et al. explained that through subgroup analysis of student employment status, i.e., whether they worked or did not work throughout the school year, the latter were similarly more likely to analyze an increase in empathy with time, providing additional support for the moderating influence of health care experience.29 Furthermore, Reed et al. confirmed that there might be chances to tailor training to students' prior experience or levels of fundamental empathy. Teaching that aims to foster empathy might focus on individuals who are most in need given the growing number of abilities that pharmacy schools are expected to cultivate in student pharmacists (although time and resources are limited).\n\nA cross-sectional study on HIV-stigma was undertaken by Sianturi et al. in 2022, using pharmacists and pharmacy students as participants. The findings indicate that pharmacists were more knowledgeable about HIV therapy and shown greater empathy than pharmacy students. These discrepancies in knowledge and empathy may be brought on by the fact that many pharmacy students lack any prior patient-care experience.\n\nOther\n\nOther determinants that discussed in the included studies were career preference,30,31 intercultural sensitivity,26 stigma,33 altruism, grit, self-awareness,29 marital status, and family income.35\n\nIn the context of pharmacy, empathy may also differ based on career preference. Two studies discussed career preference, and both reported that in terms of students' preferred future careers, there were no significant differences in empathy.30,31 Future research should clarify whether pharmacists' empathy levels vary depending on their professional field, even though no significant mean differences were found between pharmacy students' preferred future careers in this current study.\n\nA cross-sectional study on HIV-stigma among healthcare professionals was undertaken by Sianturi et al. in 2022, using both pharmacists and pharmacy students as participants.33 This stigma has been characterized as the irrational belief, unfavorable behavior, and negative attitude toward patients due to their HIV status. The findings indicate that compared to students, pharmacists demonstrated greater empathy and knowledge about HIV treatment. Many pharmacy students lack patient care experience, which could account for the disparities in knowledge and empathy. Lower levels of empathy and inadequate HIV knowledge were substantially associated to stigma.\n\nAltruism, grit, and self-awareness were additional factors that were perceived to be related to empathy scores. According to Reed et al. there is a weak to moderate correlation between empathy and altruism. Along with grit, empathy was tangentially associated with neither self-awareness nor locus of control.29 Ekong et al. showed that the Pearson correlation demonstrated a strong association between higher attitudes toward empathy and a higher propensity for intercultural sensitivity. This relationship was like that with intercultural sensitivity.26 On the other hand, to Fashami et al. found no statistically significant variations were found between empathy scores and its dimensions with regard to marital status and family wealth, in their 2021 study.35\n\n\nDiscussion\n\nThe objective of this systematic review was to identify and assess the determinants related to pharmacy students' empathy. This review revealed a clear trend between studies on the factors that related to the empathy of pharmacy students, despite variations in study design, data collection, and the results.\n\nOne determinant from this review that deserves attention is that females appear to exhibit a higher level of empathy than males. Some included studies stated that female students had a higher level of empathy than male students, while other studies explained that there was no discernible difference between the two groups in terms of empathy. According to Klein & Hodges, there are no aptitude differences between males and female that could account for gender disparities in empathy levels.37 They contend that practically everyone may develop greater empathy if they are given the right motivation. While Bratek et al. evaluated the level of empathy among medical school students, trainees, residents, and specialists, the results revealed that female participants had greater empathy scores than male participants.38 The existence of sex-based differences in the levels of empathy displayed by healthcare personnel indicates the potential benefits of targeted reinforcement of the honed abilities for empathetic responses during patient-centered communication skills training.\n\nOther sociodemographic factors identified were the year of study and type of school. Our findings revealed that while some studies reported that there was no significant difference between the study years, others claimed there was a considerable increase in empathy in the fourth year compared to the prior year. These findings are comparable to those from Magalhães et al., who found that sixth-year students scored more highly on empathy scores than first-year medical students.39 They raise the prospect that restrictions might have been raised during medical training. These variations may be caused by different levels of clinical exposure throughout undergraduate courses, different teaching strategies, or cultural variations.32 The type of school is also associated with the level of empathy among pharmacy students. According to one of the included studies, students in women's universities showed more overall empathy than those at co-educational universities.30 This may be connected to the instructional environment at each school, or the social environment.\n\nBeyond the determinants described above, educational interventions, experience, and career preference also exert an influence on pharmacy students’ empathy. The result suggest implementation of educational intervention in the institution's curriculum is crucial. According to a systematic review by Batt-Rawden et al., educational interventions can be successful in upholding and fostering empathy in undergraduate medical students.3 Furthermore, they emphasize the requirement for multicentre, randomized controlled trials that disclose long-term data to assess the durability of intervention effects. Similarly, Pratiwi et al. showed that educational interventions can successfully encourage empathy in pharmacy students. The majority of the study that is included use experiential training techniques like simulations, role-playing, or learning based on conceivable scenarios or games to increase participants' empathy.40 The study of health professional students’ empathy levels enables us to determine whether it can be used to predict the choice of university course and whether it is a stable quality or changes during the course curriculum. As an example, research done by Saiva et al. in 2020 suggests simulation may be a useful educational tool to include in health curriculum. According to the JSE score, this study discovered a considerable improvement in empathy for the elderly population following the simulation.41\n\nIn the context of pharmacy, the studies reviewed argue that empathy may also differ depending on the preferences for future career characteristics. For instance, pharmacists working in community pharmacies may exhibit more empathy than those who work in hospitals because they have more opportunities to interact directly with patients.30 Our review also demonstrated how the experience has a significant impact on pharmacy students' capacity for empathy. This was also covered by Tisdale et al., who found that medical students' empathy levels considerably increased right away following their patient interaction experience and that this the persisted for five weeks.42 This is similar to findings in Boarman et al., which described involvement in geriatric experience and resulted in a statistically significant improvement in pharmacy students’ empathy scores toward geriatric.43 Additionally, survey findings show that encounters with elderly patients at a single event improved students' comfort levels during screening, counseling, and communication.\n\nThis review successfully pinpoints factors or determinants that are associated with pharmacy students' empathy. However, it has some limitations. We acknowledge that the included studies are heterogeneous, indicating that additional studies are necessary before reaching any firm conclusions. Second, because most of the studies that were included measured empathy using self-reports, they did not necessarily accurately capture the empathy of pharmacy students and how patients felt. Understanding how the patient feels and the patient's situation is critical for pharmacy students to grasp the significance of empathy for patients. It is highly advised to conduct qualitative research to obtain a more accurate understanding of the patient's feelings and empathy. Higher levels of evidence are also required in studies to address the potential bias caused by the use of self-report questionnaires, as well as other potential biases and inaccuracies. Thirdly, considering articles written in various languages would have stimulated new ideas about how pharmacy students’ differences affect empathy levels. More research is needed to properly understand the ways by which empathy levels can be improved and to develop the most effective pharmacy educational strategies.\n\n\nConclusions\n\nThis review examined 14 papers that reported on determinants connected to pharmacy students' empathy. According to the findings of this systematic review, a variety of determinants, including educational intervention, experience, gender and/or sex, type of school, year of study, intercultural sensitivity, career preference, altruism, grit, self-awareness, marital status, and family income, can all have a positive impact on increased empathy among pharmacy students. This review came up with a few suggestions for future research projects. A deeper investigation into the effects of educational interventions, experience, gender, school type, year of study, intercultural sensitivity, career preference, altruism, grit, self-awareness, marital status, and family income is recommended. The establishment of educational practices for the development of empathy in teaching and professional settings is necessary taking into account various determinants that are relevant with empathy, especially for pharmacy students who will be in direct contact with their patients.",
"appendix": "Data availability\n\nAll data underlying the results are available as part of the article and no additional source data are required.\n\nRepository: PRISMA flow chart for “Pharmacy students’ empathy and its determinants: a systematic review. https://doi.org/10.6084/m9.figshare.21259527.v1.\n\nRepository: PRISMA checklist for “Pharmacy students’ empathy and its determinants: a systematic review. https://figshare.com/articles/dataset/Untitled_Item/21458382?file=38528501.\n\nRepository: Joanna Briggs Institute Critical Appraisal Checklists according to the methodological design of the studies. https://figshare.com/articles/dataset/Critical_Appraisal/21455916.\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).\n\n\nReferences\n\nHojat M, Gonnella JS, Nasca TJ, et al.: Physician empathy: definition, components, measurement, and relationship to gender and specialty. Am. J. Psychiatry. 2002; 159(9): 1563–1569. 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PubMed Abstract | Publisher Full Text | Free Full Text\n\nSimko LC, Rhodes DC, Gumireddy A, et al.: Effects of a Chronic Pain Simulation Empathy Training Kit on the Empathy of Interprofessional Healthcare Students for Chronic Pain Patients. Clin. Simul. Nurs. 2021; 56: 66–75. Publisher Full Text\n\nReed BN, Haines ST, Holmes ER: The Impact of Two Longitudinal Professionalism Courses on Student Pharmacists’ Empathy. Am. J. Pharm. Educ. 2021; 85(2): 8083–8130. Publisher Full Text\n\nJeon S, Cho E: Assessment of Korean Pharmacy Students’ Empathy Using the Jefferson Scale of Empathy. Am. J. Pharm. Educ. 2015; 79(5): 67. PubMed Abstract | Publisher Full Text | Free Full Text\n\nLi L, Wang J, Hu XM, et al.: Empathy In Chinese Pharmacy Undergraduates: Implication for Integrating Humanities Into Professional Pharmacy Education. Indian J. Pharm. Educ. Res. 2015; 49(1): 31–39. Publisher Full Text\n\nHall M, Hanna LA, Hanna A, et al.: View of Empathy in UK pharmacy students: assessing differences by gender, level in the degree programme, part-time employment and medical status. FIP Pharmacy Education;2015. Accessed August 1, 2022.Reference Source\n\nSianturi EI, Latifah E, Pane M, et al.: Knowledge, empathy, and willingness to counsel patients with HIV among Indonesian pharmacists: a national survey of stigma. AIDS Care. 2022; 34(1): 21–28. PubMed Abstract | Publisher Full Text\n\nFong Z, Lee S, Yap K, et al.: Impact of an aging simulation workshop with different debrief methods on the development of empathy in pharmacy undergraduates. Curr. Pharm. Teach. Learn. 2021; 13: 683–693. Accessed October 5, 2021. PubMed Abstract | Publisher Full Text Reference Source\n\nMirzayeh Fashami F, Nili M, Mottaghi M, et al.: Measuring Empathy in Iranian Pharmacy Students: Using The Jefferson Scale of Empathy-Health Profession Students. Am. J. Pharm. Educ. April 25, 2022; 8687. PubMed Abstract | Publisher Full Text\n\nWilson SE, Prescott J, Becket G: Empathy levels in first- and third-year students in health and non-health disciplines. Am. J. Pharm. Educ. 2012; 76(2): 1–4. PubMed Abstract | Publisher Full Text | Free Full Text\n\nKlein KJK, Hodges SD: Gender Differences, Motivation, and Empathic Accuracy: When It Pays to Understand. Vol. 27. University of Oregon;2001; pp.720–730. Publisher Full Text\n\nBratek A, Bulska W, Bonk M, et al.: EMPATHY AMONG PHYSICIANS, MEDICAL STUDENTS AND CANDIDATES. Psychiatr. Danub. 2015; 27: 48–52.\n\nMagalhães E, Salgueira AP, Costa P, et al.: Empathy in senior year and first year medical students: A cross-sectional study. BMC Med. Educ. 2011; 11(1). PubMed Abstract | Publisher Full Text | Free Full Text\n\nPratiwi H, Kristina SA, Widayanti AW, et al.: Educational Interventions to Improve Pharmacy Students’ Empathy Towards Geriatrics: A Systematic Review. Indian J. Pharm. 2022; 33(2): 174–185. Publisher Full Text\n\nSaiva A, Abdool P, Naismith L, et al.: An Immersive Simulation to Build Empathy for Geriatric Patients with Co-Occurring Physical and Mental Illness. Acad. Psychiatry. Springer;2020; 44. : 745–750. Accessed October 5, 2021. PubMed Abstract | Publisher Full Text\n\nTisdale CE, Black AC, Jain S, et al.: The Impact of Meeting Patients with Neurological Disorders on Medical Student Empathy. Med. Sci. Educ. 2020; 30(4): 1561–1568. PubMed Abstract | Publisher Full Text | Free Full Text\n\nBoarman EA, Nisly SA, Martin D: Use of a health screening and education event to change student attitudes toward the elderly. Curr. Pharm. Teach. Learn. 2017; 9(1): 101–107. PubMed Abstract | Publisher Full Text"
}
|
[
{
"id": "162817",
"date": "03 Mar 2023",
"name": "Ahsan Saleem",
"expertise": [
"Reviewer Expertise Pharmacy practice"
],
"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 giving me the opportunity to review this manuscript. It’s a systematic review focused on empathy and its determinants in pharmacy students. I have provided my specific comments to improve the quality of the manuscript.\nI have spotted several grammatical and linguistic errors throughout the manuscript; therefore it needs a copy edit.\n\nEmpathy is not something that can be possessed. It can be developed or enhanced, but not possessed. Please rephrase “Empathy for others must be possessed by healthcare professionals and healthcare students as healthcare professionals’ candidates, including the pharmacy student” to “Healthcare professionals and students must have or develop empathy for others.”\n\nPlease remove ‘they’ from “but they also result in favorable patient outcomes such as better patient self-care, higher patient satisfaction and faster recovery times.”\n\nPlease rephrase this sentence “We used peer-reviewed original papers, full text, must assess determinants that are associated with pharmacy students' empathy, and only be focused on pharmacy students (first to the fourth year) as healthcare professionals candidates.”\n\nThe aims mentioned in the abstract are not aligned with the main text of the manuscript.\n\nDatabase search was only limited to PubMed, Science Direct, and Scopus. I am wondering why International Pharmaceutical Abstracts was not searched given that the focus of the review is on pharmacy professionals and students.\n\nWhy search duration was limited from July 14, 2022, to July 16, 2022. Is that the date when a search was conducted? What was the actual search duration?\n\nWhat is publication age? Are you referring to the date of publication?\n\nThe findings of this systematic review have been reported under several themes, but I could not find how the authors did the thematic analysis. What was the method used to generate themes?\n\nAre the rationale for, and objectives of, the Systematic Review clearly stated? Yes\n\nAre sufficient details of the methods and analysis provided to allow replication by others? Partly\n\nIs the statistical analysis and its interpretation appropriate? Partly\n\nAre the conclusions drawn adequately supported by the results presented in the review? Yes",
"responses": []
},
{
"id": "199445",
"date": "25 Aug 2023",
"name": "Logan Thomas Murry",
"expertise": [
"Reviewer Expertise Pharmacy Student Development",
"Qualitative Methods",
"Empathy",
"Community Pharmacy Service Evaluation",
"Pharmacy Education",
"Continuing Professional Development",
"ACPE Standard 4 Skill and Key Element Acquisition"
],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 the manuscript “Pharmacy students’ empathy and its determinants: a systematic review.” The discussion of determinants is robust and well-detailed. I have concerns related to the objectives and the study eligibility criteria, specifically why other reviews and qualitative studies were excluded. Below are specific comments and suggestions related to components of the manuscript.\nAbstract:\n\nFor methods, it might be helpful to include some additional methods reflecting the search strategy such as the dates, specific keywords or MESH terms. This would allow readers to know what period articles were include from and how the total number of articles was identified.\nFor results, including the total number of articles identified would help to understand the number of articles which met inclusion criteria.\nBackground\n\nThe background generally provides good rationale for the value added by this study.\n“significance of increasing empathy in health professions students.” Is this intended to mean “despite the importance and increased attention to fostering empathy in health profession students.” ?\n“As personal discomfort from burnout, sadness, and diminished quality of life develops among trainees during their training, they are less likely to feel or show empathy. Neumann et al. found that deficits in the formal (e.g., lack of formal empathy training), informal (inadequate mentors and improper learning environments), and factors outside of the medical curriculum (e.g., abuse of students and high workload) medical curriculum.” Medical curriculum repeated twice here.\nThe objectives of the study are listed as answering two questions, the first being: 1) How is empathy measured? The title and background place most of the emphasis on determinants of empathy. Additionally, inclusion criteria included later potentially limit articles related to empathy measurement in pharmacy. It may be more appropriate to remove this objective as empathy measurement is not a focus of the methods.\nMethods\n\nAs mentioned above, I have concerns related to the study objectives and in the eligibility criteria for studies. Additionally, what justification exists for the exclusion of review articles and qualitative studies? Qualitative studies are often useful in developing theoretical frameworks, which would help understand antecedents or contributing factors/domains to empathy development.\nThe article assessment and data extraction process are seemingly robust and well-described.\nResults\nThe description of determinants related to pharmacy student empathy was very well done and thorough.\nThe section “Outcome Measures” appears to address the first object, How is empathy measured? Importantly, the review identified all articles used self-reported measures. This is likely a result of the inclusion criteria, as other studies have assessed empathy without quantitative self-report measures.\nDiscussion\n\n“Additionally, survey findings show that encounters with elderly patients at a single event improved students' comfort levels during screening, counseling, and communication.” This statement appears to be missing a citation.\n“Second, because most of the studies that were included measured empathy using self-reports, they did not necessarily accurately capture the empathy of pharmacy students and how patients felt. Understanding how the patient feels and the patient's situation is critical for pharmacy students to grasp the significance of empathy for patients. It is highly advised to conduct qualitative research to obtain a more accurate understanding of the patient's feelings and empathy.”\nQualitative research on patient feelings and empathy could be included in this review, as there are published qualitative studies looking at empathy. The inclusion of predominately self-reported assessments are potentially a result of the eligibility criteria.\nConclusions\n\nConclusions are well-written and are appropriate for the 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? Not applicable\n\nAre the conclusions drawn adequately supported by the results presented in the review? Yes",
"responses": []
},
{
"id": "199443",
"date": "31 Aug 2023",
"name": "Fatemeh Mirzayeh Fashami",
"expertise": [
"Reviewer Expertise I am pharmacist (Pharm.D) with Master of science in Health research methodology and prior experience in measuring empathy among pharmacy students. My article is cited in this manuscript."
],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThank you for giving me the opportunity to review this manuscript.\n\nBackground:\n1. You did not answer the question, “How is empathy measured?” You answered, “How is empathy measured in pharmacy students” Please add that.\n2. I think it would be valuable to add data regarding the most common tools to measure empathy in pharmacy students in the introduction.\nMethod:\n3. This is your statement:\nThe retrieved studies from databases were screened for their title and abstracts by three authors (HP, SAK, and AWW) to ensure they fit the eligibility criteria.\nIt is not clear if the reviewers reviewed each article independently or not. Please indicate.\nAccording to the PRISMA checklist: Specify the methods used to collect data from reports, including how many reviewers collected data from each report, whether they worked independently, any processes for obtaining or confirming data from study investigators, and, if applicable, details of automation tools used in the process.\n4. Your last search was one year ago. You need to update your search and include the studies that were published from 2022 to 2023.\n5. What is your justification for only including 1st to 4th-year students? Please indicate that. Based in different countries, there are bachelor's and master of pharmacy or pharm.D degrees. In some countries, both master's and pharm.D degrees exist, but in some others, only one of them is applicable. By excluding by years in pharmacy programs, what will happen to the countries that have 6-year pharm.D programs? (Like Iran) or what will happen to the students who are in 4-year programs but have delays in graduation and are now in 5th year?\n6. Generally, you need to talk about the questionnaires and how empathy is measured. JSE is a standardized tool for measuring empathy, and you need to explain that.\nResults:\n7. PRISMA diagram: You should describe the PRISMA diagram according to your defined PICO. Instead of reviewing articles and indicating design, you can combine all the non-pharmacy students in one category. Also, you should indicate why 101 hits were not relevant (due to population, design, or outcome…)\n8. Related paragraph:\nThe study titles and abstracts were reviewed according to inclusion criteria i.e., they must be focused on pharmacy students (first to the fourth year) as healthcare professionals’ candidates, and they must assess determinants that are associated with pharmacy students’ empathy. In cases where there was uncertainty regarding whether the title and abstract adequately expressed the determinants related to pharmacy students’ empathy, the full text was read. Disagreements among the reviewers (HP, SAK, and AWW) on whether the title and abstract met the inclusion requirements were settled through discussion until consensus was reached. We held discussions by getting together and going over titles and abstracts that would meet our inclusion criteria. We read and discussed the entire article together, considering the key points for entering the included studies until we get to an agreement if an article is identified that is doubtful considering the inclusion criteria.\nThis part is not relevant to the results. Move it to method. Also, there are repetitive parts in results that are focused on the method section. You have to move all of them to method and ONLY talk about results. The PRISMA diagram is presented to prevent explaining too much in the results section.\n9. Reword this section: Out of these, 120 items were excluded at this stage because they failed to fulfill the requirements for inclusion, including review articles, publications with populations of non-pharmacy students, and articles that were not pertinent to our topic. For example, several studies focus more on nursing students,18–20 another study focuses more on dietetic students.21\nOut of X number of hits from databases and grey literature search, Y were excluded mainly due to duplicate (n1), study design (n2), different population (n3) …\n10. In Table 1, you indicated that Fashami et al reported no significant changes in empathy score and pharmacy year. In the test, you have mentioned that this study did not include education level. This reference included the educational level of students, and you can see that in “Table 1. Demographic Characteristics of the Study Population”. Please revise this section and all the related parts.\n11. In this paragraph, A total of four studies of included studies evaluated the association between experience and the level of empathy. You need to specify what experience is. You are reviewing pharmacy students’ empathy, and usually, pharmacy students are not allowed to work until the end of their studies. What experience do you mean? You need to explain your experience in the method section.\n12. In the results, you have a subheading as Other. It is not a suitable subheading. Revise it.\nDiscussion:\n13. In this sentence: According to Klein & Hodges, there are no aptitude differences between males and females that could account for gender disparities in empathy levels.37 They contend that practically everyone may develop greater empathy if they are given the right motivation.\nThis sentence is non-relevant to assessing gender and empathy. You should remove or substitute is with a reference related to gender.\n14. In the 3rd paragraph of the discussion, you are discussing the relationship between study year and empathy. You discussed 4th-year students, but you should add some explanation about your findings about 2nd-year students with lower empathy scores as well.\nAlso, among all the studies that assessed school year and empathy score, many had no differences between empathy scores in different years. Do you still think that it is important? Why did you not talk about sample size here? Maybe studies with bigger sample sizes showed no effect or effect. You need to discuss your findings. Otherwise, a narrative review that neglects many non-significant differences is worthless. What if you had 20 studies and only 1 showed school grade is a predictor? Do you neglect those 19 studies?\nI suggest that you should at least add sample size to the cumulative studies that showed no difference versus the ones that showed the difference. In addition, you need to pay attention to the countries. You have many studies from the US, do the results from the US have homogeneity?\n15. In limitation: You stated that: We acknowledge that the included studies are heterogeneous, indicating that additional studies are necessary before reaching any firm conclusions.\nHeterogeneity does not always come from the limited number of studies. It can be due to the cultural differences and the pressure that students experience during their studies. You have to discuss all the possible reasons in the discussion so that in the limitation part, your audience can accept this reason from your side.\n16. In this sentence: Thirdly, considering articles written in various languages would have stimulated new ideas about how pharmacy students’ differences affect empathy levels.\nWhat do you mean by articles written in various languages? Did you include non-English articles? Or you want to indicate different languages in the empathy questionnaire. You should be cautious here because authors have to use a validated version of tools in a second language to make sure the meaning of the tools has not been changed. Checking JSE tools for empathy, you can see that validated questionnaires exist in different languages.\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? No\n\nAre the conclusions drawn adequately supported by the results presented in the review? Partly",
"responses": []
},
{
"id": "159584",
"date": "13 Sep 2024",
"name": "Tuangrat Podha",
"expertise": [
"Reviewer Expertise Health economic",
"Health service research",
"Health policy"
],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis manuscript is quite complete in providing the detail of a comprehensive review which allow replication by others.\nThe finding from systematic review of Pharmacy students' empathy and its determinants could be generalized to other contexts.\nThe finding of this study could be generalize to other context so that the other researchers who are interested in measuring and developing empathy in health science studies can learn from this study. The conclusion of the results is clear and useful for the development of empathy in teaching of the pharmacy students those will be an important part of healthcare team.\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": []
}
] | 1
|
https://f1000research.com/articles/12-18
|
https://f1000research.com/articles/9-1448/v1
|
11 Dec 20
|
{
"type": "Research Article",
"title": "Experiences of the International In-Training Examination (I-ITE) by Rwandan pediatric residents – a mixed-methods description of candidate feedback",
"authors": [
"Peter Thomas Cartledge",
"Christian Umuhoza",
"Natalie McCall",
"Christian Umuhoza",
"Natalie McCall"
],
"abstract": "Background: The University of Rwanda is the only African residency to have implemented the pediatric International In-Training Examination (I-ITE) as a tool to monitor resident knowledge acquisition. The objective of this study was to better understand the acceptance and relevance of this exam to residents from this setting and their perceptions regarding this assessment tool. Methods: This is a mixed-methods study describing candidate feedback. Immediately on completing the I-ITE residents provided feedback by filling in an electronic questionnaire comprised of four closed Likert questions and an open text box for free-text feedback. Participants were pediatric residents from the University of Rwanda, the only university in Rwanda with a pediatric residency program. Quantitative analysis of the Likert questions was undertaken descriptively using SPSS. Free-text feedback was coded and analysed. No specific guiding theory was used during the qualitative analysis, with coding and analysis undertaken by two researchers. Results: Eighty-four residents completed a total of 213 I-ITE sittings during the five exam cycles undertaken during the study period. Quantitative and qualitative feedback was given by residents during 206 and 160 sittings, giving a response rate of 97% and 75%, respectively. Five themes emerged from the qualitative analysis; 1) undertaking the I-ITE was a positive experience; 2) exam content; 3) formative nature of the assessment; 4) challenges to completing the exam; 5) practicalities to undertaking the exam. Conclusion: Qualitative feedback demonstrates that the I-ITE, a standardized, and independent exam, produced by the American Board of Pediatrics, was valued and well accepted by Rwanda pediatric residents. Its formative nature and the breadth and quality of the questions were reported to positively contribute to the residents' formative development.",
"keywords": [
"In-training Examination",
"Formative Feedback",
"Medical Education",
"Global Health",
"Rwanda",
"Internship and Residency",
"Feedback",
"Perception",
"Qualitative"
],
"content": "Abbreviations\n\nABP: American Board of Pediatrics; CBA: computer-based assessment; HRH: Human Resources for Health; I-ITE: International In-Training Examination; IRB: institutional review board; ITE: In-Training Examination; LMICs: low- and middle-income countries; MCQs: multiple-choice questions; PBA : paper-based assessments; PI: Principal Investigator; UR: University of Rwanda; US: United States.\n\n\nIntroduction\n\nFor four decades, pediatric residents in the United-States (US) have been given an annual In-Training Examination (ITE) as a formative self-assessment instrument1,2. The objective of the pediatric ITE is to assess general pediatric knowledge and to track progress year-to-year while being able to compare individual scores with national peers. Research has shown that the ITE examination is a valid and reliable estimate of resident knowledge and can predict final performance in the pediatric board examination2–4.\n\nThe I-ITE is offered annually by the American Board of Pediatrics (ABP) to training institutions globally to assess core areas of general pediatric knowledge and to gauge trainee knowledge acquisition from year to year. The content of the I-ITE is a subset of 150 to 200 multiple-choice questions (MCQs) from the US board certification exam, which is formulated by 30 board-certified experts4. The I-ITE is annually reviewed by physicians (a reviewer and 1-2 medical editors), in an attempt to ensure current/relevant information, and, where possible, to remove any “Americanisms”, terminology, etc. The I-ITE was piloted with one training program in Lebanon in 2008, and made available to all countries in 20095. The I-ITE has been shown to be a tool that can measure resident knowledge acquisition and institutional factors supporting residents in a resource-limited country, but has not be studied otherwise.\n\nIn 2013, 23 African countries were offering a postgraduate training program in pediatrics6. In Rwanda, there is a single pediatric residency program graduating approximately five to 18 pediatricians per year. Rwanda is the first sub-Saharan nation to employ the pediatric I-ITE to provide formative feedback to individual residents and to give feedback to the faculty on the overall performance of residents within the residency program5. The I-ITE, however, is a formative assessment based on the American ITE examination, and though adapted it is being utilised in a very different cultural and medical context in terms of both the populations, patients and pathologies encountered, but also in respect to the language skills, learning styles and asseessment experience of the residents undertaking the assessment. There is currently no literature regarding the acceptance and relevance of this exam to residents from this setting and their perceptions regarding this assessment tool.\n\nThis study sought to describe the experiences, satisfaction and acceptability of this online formative assessment within the specific context of Rwandan residents by identifying themes within the written feedback given by Rwandan residents who had taken the I-ITE between 2013 and 2018.\n\n\nMethods\n\nEthical approval was gained from the Institutional Review Board (IRB) of the University of Rwanda (UR). Ref: 202/CMHS_IRB/2019. Participation in the exam and giving feedback was deemed as consent, with this consent process having been reviewed and approved by the UR IRB. No personal resident data was used in the analysis. Names were removed and replaced with unique identifiers. No significant physical, social, emotional, legal and/or financial risks to participants were identified.\n\nWe undertook a retrospective mixed-methods study to describe candidate feedback. The reporting of this qualitative study has been verified in accordance with the COREQ checklist for qualitative studies (see Reporting guidelines7)8,9.\n\nUR, the only university in Rwanda with a pediatric residency program. Residents are trained at four tertiary level teaching hospitals in Rwanda.\n\nWe sought to gain a rich and detailed understanding of the experiences, attitudes and perceptions of the residents, and for the results to emerge from the data. Our previous paper has demonstrated that the I-ITE exam is a valuable tool to measure knowledge acquisition in Rwandan residents5, whereas this current paper, using qualitative methods, gave the opportunity to understand the residents’ experience and the cultural relevance to them as end-users.\n\nThe I-ITE was implemented into the UR pediatric residency program from 2012 to 2018 by the faculty of UR and the Human Resources for Health (HRH) program5,10,11. A full description of the implementation of the I-ITE in Rwanda, and the performance of the residents in this assessment, has been previously described in the literature5,12.\n\nThe I-ITE is an assessment employing single best answer MCQs, executed electronically (Figure 1), requiring each participant to have access to a computer, usually a laptop, and internet access. The exam is undertaken in a quiet room, proctored by a faculty member. On completing the MCQ exam, it is routine ABP practice for residents to provide feedback by filling in a digital/electronic questionnaire comprised of four closed Likert questions and an open text box for free-text feedback (Figure 2). All questions and feedback were provided in English, the official academic language of Rwanda. The investigators of this study were not involved in the design of the questions used in the feedback as these were standard questions used by the ABP. Completing the Likert questions and free-text comments were not obligatory to complete the I-ITE. The Likert scores and feedback comments were provided by the ABP.\n\nAll pediatric residents enrolled in the residency program from 2012–2018 undertook the I-ITE formative assessment. Pediatric residents in Rwanda have completed undergraduate medical school and a minimum of one year as a general practitioner in a district hospital. No sampling methods were used as all resident I-ITE exam sittings were used in the analysis.\n\nThe primary objective of implementing the I-ITE was for the formative benefit of the residents. No other incentives were offered to residents to undertake the I-ITE exam or give feedback and participants enrolled in the I-ITE voluntarily.\n\nAll residents in the program undertook the I-ITE under the period of study, and all data were analyzed; therefore, no sample size calculation was performed, and saturation was not considered.\n\nQuantitative data are reported using descriptive statistics, using Statistical Package for the Social Sciences (SPSS)13.\n\nA pragmatic approach was taken. No specific guiding theory was used.\n\nFree-text comments were submitted electronically by residents immediately after completing each exam sitting; therefore, no transcription was required. Transcripts were not returned to participants for comment or correction. The data was coded by the Principal Investigator (PI, PC), in Microsoft Excel (V16.17.27), with codes identified de novo, as they developed14. Nvivo (V11.4) was used to create a word cloud, combining stemmed words. No codebook was created prior to starting the analysis. Coding was then double-checked by a second investigator (NM) with amendments made.\n\nAn inductive approach was taken to analysis. No hypothesis was formed prior to the research. The aim of the study was to simply describe the lived experiences of the residents in this setting. This study employed conventional content analysis. The goal of the content analysis was to “provide knowledge and understanding of the phenomenon under study”15. Themes were not identified in advance. Thematic content analysis was performed by the PI undertaking the following steps16: i) familiarization of the data; ii) identifying codes and themes found in the transcripts by searching for repetition, metaphors, analogies, and “in vivo” categories used by participants; iii) organizing codes and themes for presentation. Participants did not provide feedback on the coding or thematic analysis. The Likert questions allowed for a degree of triangulation of the qualitative data and to assess the trustworthiness of the written, free-text, comments.\n\nPC is a male, British pediatrician; NM is a female, Swiss-American pediatrician; and CU is a male, Rwandan pediatrician. At the time of the study, PC and NM were working in Rwanda on the Human Resources for Health (HRH) program, and CU is a member of faculty at the University of Rwanda. All investigators are clinicians specializing in pediatric medicine and medical education. The investigators are responsible for supervising UR pediatric resident research activity and collectively have experience undertaking and supervising qualitative research activities.\n\nAll three investigators have professional relationships with the residents who were participants in the study. The data was removed of personal identifiable details to minimize bias of the comments made. Though data collection was prospective with feedback provided immediately after the completion of the exam, the analysis of the feedback was retrospective. Therefore, participants were unaware of the personal goals and reasons for doing the research at the time of completing the feedback questionnaire.\n\nAs the feedback was provided digitally and in English this may have biased the feedback to participants who possess better English language skills and those with better IT skills. The data has the potential to be biased by social desirability bias. All three investigators were members of the pediatric residency faculty and therefore, may have held biases and assumptions regarding the I-ITE before undertaking the analysis. No formal steps were taken to measure or alter these biases.\n\n\nResults\n\nNo data-points were missing in the data provided by the ABP.\n\nEighty-four residents completed a total of 213 I-ITE sittings during the five exam cycles undertaken during the study period. The I-ITE exams were undertaken by residents in November 2012 (n=27), March 2014 (n=41), April 2016 (n=50), March 2017 (n=49) and May 2018 (n=46). Demographic data (age, etc) is not requested in the feedback tool and therefore is not available.\n\nThe Likert questions were completed at 206 of the 213 exam sittings, giving a response rate of 97%. Residents found the online testing system easy to use and felt that the examination was a good formative assessment of their knowledge in general pediatrics (Table 1). They were more undecided regarding the difficulty of the exam in terms of their level of training. Overall, residents scored that the items were not culturally appropriate for the practice of general pediatrics in Rwanda, though the most common (modal) response of residents were undecided on this question.\n\nAll the comments were made in English. Meaningful free-text feedback was given following 160 exam sittings, giving a response rate of 75%. Median comment length was 21 words long (min=2, max=86). Five themes emerged from the analysis (Table 2). Nvivo was used to produce a word cloud to visualise the commonly used words in the participant feedback (Figure 3). The sex of the participant and the year of the exam sitting are provided with all the quotes below.\n\nTheme 1: I-ITE as a positive experience\n\nResidents consistently reported that undertaking the I-ITE was a positive experience. Codes within this theme were the most commonly coded items during the analysis. Residents valued the experience, the quality of the assessment, and reported that it was suited to their level of training,\n\n“This was a good experience” [F, 2015],\n\n“This exam was incredible. I really appreciated how it was set” [M, 2013]\n\n“This was a really good assesment for my level of training”[M, 2016].\n\nThis high level of end-user satisfaction is an important aspect of formative assessments to ensure that residents remain engaged in the process. This is especially important where the I-ITE is offered annually during a four-year program.\n\nTheme 2: Exam content\n\nSubtheme: Pathology from developed nations\n\nForty-nine residents commented that the questions tested unfamiliar pathologies, investigations, and terminologies.\n\n“… sit and ask yourselves if knowing American stuff like - Little league - Gun induced shot etc. These are for Americans not for Africans. For us, we deal with malaria, diarrhea, pneumonia and other tropical illnesses” [M, 2017].\n\nThe MCQ questions used in the I-ITE are not explicitly written for settings such as Rwanda. This theme was supported by the Likert questions, suggesting that residents did not feel that the questions were culturally or epidemiologically appropriate.\n\nSubtheme: Pathology from resource-limited nations\n\nSimilar to the presence of American pathology was the lack of pathology relevant to settings such as Rwanda.\n\n“I could suggest that next time you consider also tropical medicine where malnutrition, malaria,.... are a big concern” [F, 2016]\n\nThis is an important point worth considering. However, the I-ITE is undertaken in many settings outside of the USA and is not exclusively for the use of resource-limited countries with similar pathologies to Rwanda. Therefore, tailoring it to each country is like to be beyond the scope of the ABP. In addition, the I-ITE is designed to match the ITE given in the US closely. Removing too many questions may modify the ITE to the point of being completely different and, therefore, uncomparable. These comparisons give significant meaning to the formative nature of the assessment process.\n\nSubtheme: Breadth of the pediatric curriculum\n\nFormative assessments are important to allow residents to assess their own performance in relation to that expected within a curriculum17. The Rwandan pediatric residency follows a curriculum based upon a modified, locally adapted version of the Global Pediatric Education Consortium curriculum, also developed by the ABP18. Residents reported that the exam was good at assessing the breadth of the curriculum topics of general pediatrics. This was despite the use of Americanisms and pathologies and the lack of locally relevant pathologies.\n\n“This [I-ITE] is fundamental as it provides the base line common knowledge in pediatrics” [M, 2012].\n\nThe I-ITE is a formative assessment, and as such, it gives residents a time-specific assessment of their general level of knowledge without being overly sub-specialized. Though not all topics are assessed, it provides residents with benchmarking of the areas for improvement.\n\nSubtheme: Item difficulty\n\nA subset of residents found that the questions in the exam were difficult. Reasons for this difficulty were; lack of time, language skills, the relevance of pathology, not having encountered investigations in their setting, length of text in the scenarios, being assessed early in residency, breadth of topics assessed, and the perceived level of knowledge of Rwandan residents.\n\n“this was difficult for our knowledge” [F, 2013]\n\n“many question were not appropriate for the level of my training and also many of them should be differently answered when practicing in my settings” [F, 2015]\n\nThe I-ITE scores gained by Rwandan residents were significantly below residents from other international settings5. The difficulty of questions was based on multiple factors. The comments found within this subtheme did not triangulate with the Likert question responses regarding difficulty where residents did not feel that the difficulty was excessive for their level of training. Therefore these free-text comments may have reflected the experiences of a subset of the cohort.\n\nSubtheme: High-quality assessment process\n\nThe residents reported that the assessment was of a high quality. They reported that the questions were clear and concise and that it was well structured. No residents gave any concerns regarding quality, only the relevance of questions. Comments regarding the assessment being “well prepared” may reflect the involvement of local faculty to ensure that the exam ran smoothly, rather then the I-ITE exams themselves.\n\n“The exam was well prepared and the questions were clear and concise.” [M, 2015, P47]\n\n“The exam was well structured” [M, 2017, P36]\n\n“Good and well prepared examen with easy online system.” [M, 2015, P46]\n\nThe I-ITE has undergone rigorous processes to ensure that the quality, structure and format of questions is high, the residents well appreciated this.\n\nTheme 3: Formative nature of the exam\n\nSubtheme: Self-development (formative nature)\n\nResidents valued the formative nature of the I-ITE exam. Residents felt it helped them to identify areas for personal reading and development. They also valued being able to assess their own level of performance compared to their peers. Many residents also reported that the exam acted as a motivator for future study.\n\n“Thank you for helping me to asssess my level. I discovered my weakness and I am going to improve my area of weaknesses” [M, 2016]\n\n“The examination was hard. But it reminds me to work hard” [M, 2015]\n\nMany residents had a clear understanding of the formative nature of the exam, benchmarking them against peers and identifying areas for improvement. The qualitative responses within this sub-theme were supported by the quantative responses to the Likert question, with nearly 80% agreeing that the exam was good for assessing their level of performance.\n\nSubtheme: Level of training\n\nIn the US, the pediatric ITE is used by all residents, of all levels, to benchmark themselves, throughout residency, in preparation for their pediatric board exams. Board equivalent exams are not undertaken in Rwanda. Though the feedback (above) demonstrated a degree of general understanding regarding formative assessment, it was evident that many residents had not fully grasped the concept of this formative assessment being employed as a tool to track their own progress over a series of years. For example, several residents wanted bespoke exams based on their level of training at the time of taking the exam, which would not enable a tracking of performance during residency.\n\n“It can be better if the exam is different depending on which level of the training you are” [M, 2015]\n\nThere was, however, a small sub-set of first-year residents who appreciated being able to assess their level of knowledge at this early stage in their residency.\n\n“Thank you for the examination, I am resident in first year and it helped me to know my level of weakeness so that I will improve” [F, 2016]\n\nSubtheme: Feedback on answers\n\nThere was a desire from residents to be given the answers, along with feedback, to the individual question items. This reflects the desire of residents to use the I-ITE as a tool for learning rather then an assessment of their performance.\n\n“It would be better if we get the answers to review them and improve our medical knowledge” [F, 2017]\n\nUndertaking an exam can be stressful, therefore gaining specific feedback on the questions would be helpful to residents.\n\nTheme 4: Challenges to completing the exam\n\nSubtheme: Language challenges\n\nKinyarwanda is the official, unifying language of the 12 million population of Rwanda. French, which is still an official language in Rwanda, was used in education in Rwanda until 2010. Many Rwandan residents would therefore have undertaken their primary and secondary education in French and Kinyarwanda. Language was, therefore, a challenge reported by several residents undertaking the I-ITE.\n\n“The exam contain difficult words to understand when you are not english native speaker, which make it more dificult” [F, 2016]\n\nDespite this, many residents reported that the questions were easy to read, understand and were concise.\n\n“The exam is well prepared and easy to read and understand” [M, 2017]\n\nSubtheme: Time to complete questions\n\nResidents found the experience of undertaking the I-ITE positive. However, the timing was a common challenge, with residents reporting not enough time to complete the questions.\n\n“The number of questions and length of statement/clinical scenario given with questions do not allow time for reasoning before chosing one most likely answer” [M, 2015]\n\nSubtheme: Internet connection\n\nInternet connection for residents in Rwanda is expensive, frequently not available or of low bandwidth. Internet connection was frequently described in the feedback, with many residents reporting internet connection problems during the exam.\n\n“This examination is helpful for our training in general pediatrics because it shows us our level of knowledge internationaly but it is difficult to use online system due to our slow internet connection in our setting” [F, 2017]\n\nTheme 5: Practicalities\n\nSubtheme: Preparation\n\nResidents had a desire to have sample questions with which to prepare for the I-ITE exam. They also felt that they needed more practice at MCQ format questions.\n\n“I would like to have my scoring rate and sample questions in my mail box in order to prepare the next exam.” [M, 2012]\n\nResidents in Rwanda do not commonly have access to online exam preparation materials, which are often unaffordable or not available due to internet bandwidth.\n\nSubtheme: Exam software\n\nThe online system/software used for the I-ITE were frequently discussed in the feedback. Comments were almost exclusively positive, with residents finding the system of good quality, user-friendly, and easy to use.\n\n“The online system is user friendly! The exam contained some culrurally irrelevant questions” [M, 2016]\n\nThe positive nature reported by residents was corroborated by the high Likert scores reporting that the system was “easy to use”\n\nSubtheme: Timing of taking the exam within the academic year\n\nThe timing of the exam during the academic calendar is important, with the recommendation being that it comes early in the academic year, in order to have a baseline assessment with which to have a maximum impact in the formative process for residents. The Rwandan I-ITE was often executed late in the academic year and was therefore frequently not undertaken at this optimum time due to the practicalities of enrolment and payment.\n\nInterestingly, residents reported conflicting opinions on the timing that they wished to undertake the exam in the academic calendar. Some residents wanted the exam early to give time to prepare for the end of year exam.\n\n“I also suggest that if possible this exam could be taken earlier, in the beginning, or in the middle of the academic year (december-january), as it can helps students to keep reading and prepared” [M, 2017]\n\nWhereas, first-year residents generally found undertaking the exam early in their studies a negative experience.\n\n“… and i hope that the next evaluation will be at the end of the next academic year because this one was very early only 2 months for our study …. ” [M, 2012]\n\nSubtheme: Desire to undertake I-ITE more frequently\n\nResidents reported a desire to undertake the I-ITE more frequently in order to be able to assess their level of knowledge. Different participants recommended using the exam; every three months, every trimester, or every six months.\n\n“The exam was a very good way to assess my level of knowledge. I would love to have frequent access to this examination material to train myself. Thank you!” [F, 2015]\n\nIn the US, residents undertake the ITE exam annually to gauge their preparation and readiness for the board exams. As residents in Rwanda do not take an equivalent to the board exam, their objectives to take the exam may have been different, and undertaking the exam frequently would allow them to track their performance and identify areas for improvement.\n\n\nDiscussion\n\nThis study sought to describe the experiences, satisfaction and acceptability of this online formative assessment within the specific context of Rwandan residents by identifying themes within the written feedback given by Rwandan residents who had taken the I-ITE between 2013 and 2018.\n\nResidents valued the I-ITE and reported that it was a positive experience. This was triangulated by their desire to undertake the I-ITE more frequently and their quantitative responses to the Likert questions. The feedback from our residents demonstrates that faculty from other training centers, similar to Rwanda, can confidently implement the I-ITE and that residents will positively receive this formative experience. It is important to note that this feedback was given immediately after the exam and could have been different if the feedback was given after they received their grade score. The formative nature of the I-ITE is to identify residents who may need additional support, and we did not take any steps to evaluate if residents in difficulty find that this aspect of the process causes anxiety or distress and therefore alters their perception of the exam being a positive experience. This would make an interesting piece of qualitative work in the long term. In the short term, faculty implementing the I-ITE should put systems in place to support residents when using the exam scores to identify residents who need additional support in their training.\n\nExam content. Vetting questions for quality and relevancy is critically important when creating both formative and summative assessments19,20. The residents commented on the relevancy of the questions and topics to the practice of Pediatrics in Africa and commented on the fact that there were questions about diseases and pathology which are not routinely found locally. On the other hand, residents also reported that they wish to have seen more questions about pathology more frequently seen locally, such as malaria or malnutrition.\n\nWhile developing the bank of questions for the I-ITE, the ABP had one or more medical editors to review the I-ITE to ensure current/relevant information and to remove any Americanisms before each year’s administration12. However, because the I-ITE has to be comparable to the ITE undertaken by the residents in the US, only a limited number of questions could be adapted or removed. From the residents' observations, one of our key recommendations is for the ABP to review this process carefully and to choose reviewers who have practiced outside the US to ensure that they include questions that are relevant globally, not only in the US.\n\nThe perceived difficulty of an exam can impact the evaluation of didactic teaching by learners21. More residents felt that the difficulty was appropriate (45%) compared to those who felt it was inappropriate (21%), with the remainder being non-conclusive. This was not reflected in the free-text feedback. The difficulty was reported to be related to: i) topics that were not commonly seen in Rwandan clinical practice, ii) did not correspond to the curriculum taught, and iii) the question format, which included question stems with long case descriptions and significant information to process. Some residents reported that case-based MCQ represented a new style of questions that they previously had not experienced. It is known that student’s satisfaction with MCQ exam questions can correlate with the format of the MCQ and that case-based MCQs, which measure higher reasoning skills (rather than simple memorization or recall of knowledge), can be perceived as being more difficult22,23. Our own personal experience, in Rwanda, is that official medical school and residency written examinations continue to use a higher proportion of true-false style questioning rather than a true “best-of” approach, and also more factual knowledge assessment rather than case-based reasoning skills. This questioning style could have contributed to this perception of item difficulty in the respondents.\n\nThe residents acknowledged the high quality of the tool. For formative assessment to be effective, the tool needs to be credible24. Without credibility, residents would not be able to trust the outcomes of the assessment and therefore, may be less likely to implement any learning strategies from the formative process. It is therefore very reassuring that none of the residents in our cohort expressed concerns regarding the quality of the questions themselves.\n\nOur residents found that the formative nature of the exam helped them to identify areas for personal reading and development. Research has demonstrated that the formative nature of the ITE goes beyond knowledge acquisition and improves the resident’s self-assessment skills of their own overall performance17. This is an important finding as the I-ITE not only gives them a single benchmark but provides the skills to self-assess performance and competencies in the future.\n\nOur results suggest that many of our residents did not understand some of the concepts of formative assessment tracking performance over the course of their residency. They reported a desire for an exam that was bespoke to their own level of training (e.g., as a first-year resident). This highlights the importance of preparing residents for the I-ITE to understand that it is a formative process over a number of years and that they can track their own progress and identify where their current performance measures in comparison with their peers and their previous scores. This should be done in a manner to encourage residents to take ownership of their own learning and their own formative assessment24.\n\nWhen undertaking a formative assessment, there is a natural tendency to want to understand each question and gain feedback that goes beyond a quantitative benchmark. This finding was found in residents preparing for the ITE using question format preparation materials25. However, this kind of feedback would compromise the exam bank and would not be feasible.\n\nChallenges to completing the exam. Assessing knowledge in our Rwandan residents who are not native English speakers was problematic. Though the official language of education in Rwanda is English, residents will complete any formative and summative assessments in English. The feedback identified that some residents found that completing the I-ITE in English was challenging. We, therefore, postulate that they would also have similar concerns completing their UR summative exams in English. However, it does also add to the formative nature of the I-ITE as it will have allowed residents to gain feedback on their own language comprehension in preparation for their summative assessments, undertaken in English.\n\nResidents valued the formative nature of the exam. However, this impact of a formative exam goes beyond user satisfaction. The use of an annual, formative, in-training examination in Pakistani residency programs, in combination with a number of further interventions, significantly increased the number of residents successfully completing their postgraduate examinations26. Therefore, regular formative assessment has the potential to improve long term outcomes for residents and other residency programs in resource-limited settings, such as Rwanda, may benefit from implementing the I-ITE. The benefits are to implement a high-quality formative process without requiring a substantial burden on the faculty. There are several considerations that these programs should consider when implementing the exam. Several residents reported that the amount of time available to complete each question was not sufficient. Being able to undertake questions promptly is a necessary skill when undertaking summative MCQ-style questions and is, therefore, in itself formative for the participants. After the first year of the I-ITE implementation, the amount of time given for the exam was extended. Undertaking an examination can be a stressful experience for participants; therefore, identifying methods to overcome practical challenges, such as internet connection, would likely lead to a more positive experience and more engagement with the process27.\n\nThough residents themselves did not discuss it, the I-ITE is not only formative for individual residents, but it also offers a high-quality predictor of residents in difficulty who may need early intervention to ensure they meet the required knowledge and competencies28. One residency, in the USA, implemented interventions for low scoring residents including: closer academic guidance and supervised meetings with a faculty advisor, development of a personalized study plan with an emphasis on self-study and reading, meeting with a test-taking expert, mandatory departmental didactic conference attendance and withdrawal of moonlighting privileges29. The combination of these steps was highly effective at improving resident performance. Therefore, faculties considering implementing the I-ITE should predefine the objectives of doing so, and how the strategies they will use to support residents who are underperforming.\n\nResearch has shown that residents who prepare for examinations by completing review questions result in quantitatively higher ITE scores30. It is therefore interesting to note that our residents reported a desire for sample questions with which to prepare for the exam format. The ABP, or individual departments implementing the I-ITE, may want to collaborate to create a bank of sample questions to help prepare residents for the I-ITE and other formative and summative assessments. This will give residents familiarity with the style of questions used, rather than comprehensive coverage of the pediatric syllabus. It is important to note, however, that personal study is not necessarily the optimum method for preparing for such exams, with one study finding that residents undertaking a higher number of general pediatric admissions correlated with an increase in ITE score31.\n\nIt is reported in the literature that computer-based assessments (CBA) are generally well-received for formative assessments25,32. Establishing the use of a CBA is not straightforward and can be negatively received. Implementation of a CBA requires a series of steps, namely; introducing CBA to both staff and students, using an established item bank, ensuring adequate infrastructure, developing CBA software, piloting the CBA, and making preparations for untoward incidents, and finally evaluating the exam33. The use of the I-ITE from a reputible source allowed for the majority of these steps to be undertaken without involvement of the local faculty, and we feel that this contributed to the positive satisfaction amongst our residents.\n\nOur residents have their own perceptions regarding assessment processes, which are formed within their own environment and culture. Therefore, though several important themes have been drawn from their feedback, these may not be fully transferrable to other resource-limited settings who are considering implementing the I-ITE.\n\nFuture pieces of research work may be to assess local faculty engagement and perceptions of the I-ITE qualitatively.\n\nThe single major limiting factor of the study was that the Likert and free text questions were not explicitly designed for the purpose of this study and rather designed by the ABP for all residency programs. Interviews or focus-groups could, therefore, have provided more rich data and a more comprehensive understanding of the residents' perspectives and experiences. Due to the qualitative nature of the methodology, we can only discuss what the participants reported and not what they do or did. The personal experiences and pre-existing opinions of the researchers may have biased the interpretation of the qualitative data. Just as some residents may have found it difficult to complete the I-ITE in English, these same residents may have found giving feedback in English challenging.\n\n\nConclusion\n\nWe demonstrate that the I-ITE, a standardized, and independent exam, produced by the ABP, is valued and well accepted by pediatric residents in Rwanda. Its formative nature and the breadth and quality of the questions were reported to contribute to the residents formative development positively. Residents from resource-limited settings with limited faculty and didactic teaching wish to have more frequent access to formative learning opportunities such as this type of assessment. Adapting the exam to make it more relevant to local epidemiology would be beneficial to the end-user, in their own setting and allow for a better comparison of knowledge between settings. The ABP should be applauded for producing this resource for international residencies. The low number of residencies using the I-ITE in low-income countries (LICs) should also be a point for reflection. The cost of purchasing for I-ITE is probably beyond the reach of many institutions and was only made possible in Rwanda because of the HRH programme and externally funded programme. Therefore, the ABP may wish to consider taking a HINARI approach and offer the I-ITE free of charge in LICs as a gesture of their desire to see pediatric training and practice improve in these settings.\n\n\nData availability\n\nThe datasets generated and/or analyzed during the current study are not publicly available as they are third party data owned by ABP. They can potentially be made available from the corresponding author on reasonable request for the purpose of further research. This would require providing a research protocol and consultation with the American Board of Pediatrics (data owners) and the authorizing Insitutional Review Board (University of Rwanda).\n\nHarvard Dataverse: COREQ checklist for “Experiences of the International In-Training Examination (I-ITE) by Rwandan pediatric residents – a mixed-methods description of candidate feedback”. https://doi.org/10.7910/DVN/5X5XWT7.\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\nDr. Robert Furter, Dr. Laurel Leslie, Donna Crisp (ABP, USA). Dr Cliff O’Callahan (Human Resources for Health, Rwanda)\n\nThe content of this manuscript is solely the responsibility of the authors and does not represent the official views of the American Board of Pediatrics.\n\n\nReferences\n\nThe American Board of Pediatrics: General Pediatrics In-Training Examination. Am Board Pediatr. 2018 [cited 2018 Dec 10]. Reference Source\n\nAlthouse LA, McGuinness GA: The in-training examination: an analysis of its predictive value on performance on the general pediatrics certification examination. J Pediatr. 2008; 153(3): 425–8. PubMed Abstract | Publisher Full Text\n\nGrossman RS, Fincher RME, Layne RD, et al.: Validity of the in-training examination for predicting American Board of Internal Medicine certifying examination scores. J Gen Intern Med. 1992; 7(1): 63–7. 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PubMed Abstract | Free Full Text\n\nChang D, Kenel-Pierre S, Basa J, et al.: Study habits centered on completing review questions result in quantitatively higher American Board of Surgery In-Training Exam scores. J Surg Educ. Elsevier; 2014; 71(6): e127–31. PubMed Abstract | Publisher Full Text\n\nChase LH, Highbaugh-Battle AP, Buchter S: Residency factors that influence pediatric in-training examination score improvement. Hosp Pediatr. 2012; 2(4): 210–4. PubMed Abstract | Publisher Full Text\n\nRudland JR, Schwartz P, Ali A: Moving a formative test from a paper-based to a computer-based format. A student viewpoint. Med Teach. 2011; 33(9): 738–43. PubMed Abstract | Publisher Full Text\n\nHassanien MA, Al-Hayani A, Abu-Kamer R, et al.: A six step approach for developing computer based assessment in medical education. Med Teach. 2013; 35 Suppl 1: S15–9. PubMed Abstract | Publisher Full Text"
}
|
[
{
"id": "80914",
"date": "19 Mar 2021",
"name": "Ali Dabbagh",
"expertise": [
"Reviewer Expertise Anesthesiology",
"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\nFirst of all, the authors should describe fully how they created their questionnaire; was the validity and reliability of the questionnaire measured?\n\nWhy the authors preferred to select no sample size calculation?\n\nIn the results, did they perform factor analysis?\n\nIn the discussion, is it possible to describe the results based on each level of the residents?\n\nFinally, are the quantitative results described in full? Where?\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Partly\n\nAre sufficient details of methods and analysis provided to allow replication by others? Partly\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nPartly\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes",
"responses": [
{
"c_id": "9147",
"date": "06 Jan 2023",
"name": "Peter Cartledge",
"role": "Author Response",
"response": "We would like to thank you for your helpful comments, and review. We have amended the article based on this feedback. First of all, the authors should describe fully how they created their questionnaire; was the validity and reliability of the questionnaire measured? -Response: Thank you for your feedback and comments. The questionnaire was designed by the American Board of Paediatrics. In the original manuscript, we had the text “the investigators of this study were not involved in the design of the questions used in the feedback questionnaire as these were standard questions used by the ABP.” In the revised text we have added “The questionnaire was designed by the ABP, and is not a validated tool, therefore…” prior to this to make it more clear. We have have also moved the general text about the I-ITE in general into a new subheading called “The I-ITE”. Why the authors preferred to select no sample size calculation? -Response: We did not take a sample of this population, as we used the entire cohort. As no sample was used (i.e. 100%/the full cohort) we didn’t undertake a sample size calculation, and saturation was not applicable as there were no further persons we could interview or enquire of. We have added some extra text to this section to make it a bit more clear. In the results, did they perform factor analysis? -Response: We did not undertake any factor analysis. Our data is presented in a descriptive and pragmatic approach; therefore we do not wish to over-reach what is a relatively simple descriptive analysis. In the discussion, is it possible to describe the results based on each level of the residents? -Response: We did not do this as it was not described within the objectives of the study. The reasons for this was that the questionnaires were implemented over multiple years and therefore there would be a lot of confounders that would make any analysis / interpretation difficult to undertake. Finally, are the quantitative results described in full? Where? -Response: The quantitative results are described in full in Table 1. We used these Likert question responses to triangulate the free text comments."
}
]
},
{
"id": "138352",
"date": "24 May 2022",
"name": "Gary L Beck Dallaghan",
"expertise": [
"Reviewer Expertise I am an educational psychologist with extensive quantitative and qualitative research experience."
],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 was interested to read this article to see how an American examination would translate for use in a culturally diverse part of the world. I appreciate the authors' work on this study. The following comments are intended to help them make revisions to this study.\nI'm starting with the abstract first primarily because it is the first thing one reads. After reading the entirety of the manuscript, I would argue that you did not do a mixed methods study but rather a survey study that included both quantitative and qualitative sources of data. Mixed methods requires much more deliberate planning to ensure that both quantitative and qualitative data instruments complement one another. What you did was use an established tool designed by the American Board of Pediatrics that was intended more as a feedback loop. It does not meet the criteria to classified as mixed methods. You may disagree, but I would seriously suggest you consider this.\nBecause of that, if this is intended to be survey research, you only need one response rate. That portion of your abstract was confusing to read because it is not the norm to report a response for the quantitative vs qualitative data. Since this data was submitted simultaneously, you have one response rate.\nIntroduction section read fine to me. It was very clear.\nMethods: Again, rethink this notion of mixed methods.\nIn the qualitative approach you indicate you did not use a guiding theory. However, in the thematic analysis section you reference describing the participants' lived experience. That is known as phenomenology, which is a guiding theory. I would recommend reading a bit about phenomenology and decide if that is what you should have stated. These are contradictory statements that need to be resolved.\nResults and Discussion read well to me. I did appreciate you triangulating quantitative findings with your themes.\nIt was quite interesting to read the comments from people who noted the \"Americanisms\" in the examination and how challenging that was. I also wonder if there is room for future research related to how people do based on their English proficiency.\n\nIs the work 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? No\n\nAre the conclusions drawn adequately supported by the results? Yes",
"responses": [
{
"c_id": "9148",
"date": "06 Jan 2023",
"name": "Peter Cartledge",
"role": "Author Response",
"response": "We would like to thank you for your helpful comments, and review. We have amended the article based on this feedback. Responding specifically to each comment: Comment: I'm starting with the abstract first primarily because it is the first thing one reads. After reading the entirety of the manuscript, I would argue that you did not do a mixed methods study but rather a survey study that included both quantitative and qualitative sources of data. Mixed methods requires much more deliberate planning to ensure that both quantitative and qualitative data instruments complement one another. What you did was use an established tool designed by the American Board of Pediatrics that was intended more as a feedback loop. It does not meet the criteria to classified as mixed methods. You may disagree, but I would seriously suggest you consider this. -Response: Yes, this is very reasonable and appropriate. We have changed the wording in the methods and the abstract to reflect this. We have also changed the title to reflect this. Comment: Because of that, if this is intended to be survey research, you only need one response rate. That portion of your abstract was confusing to read because it is not the norm to report a response for the quantitative vs qualitative data. Since this data was submitted simultaneously, you have one response rate. -Response: Thank you for this, and certainly changing it to make it more readable will be helpful. We’ve put a single response rate in the abstract. We’ve moved the response rate in the methodology into the participants section, to reflect a single response rate. Also, we have changed the section in the free-text (qualitative) section to represent the 160 of the 206 responders. We have also changed it in the abstract. Comment: Methods: Again, rethink this notion of mixed methods. -Response: As above. We have reworded this. Comment: In the qualitative approach you indicate you did not use a guiding theory. However, in the thematic analysis section you reference describing the participants' lived experience. That is known as phenomenology, which is a guiding theory. I would recommend reading a bit about phenomenology and decide if that is what you should have stated. These are contradictory statements that need to be resolved. -Response: Thank you, this is really helpful. As you will see from our wording regarding “lived experienced” this was something that we ourselves had difficulty fully deciding on during the design and write-up phase. Having your insight on this is helpful and we have changed the wording. We have also added the reference of “Creswell J. Chapter 4: Five Qualitative Approaches to Inquiry. In: Qualitative inquiry and research design: Choosing among five approaches. 2007. p. 53–84.” which we have used during the process. Comment: It was quite interesting to read the comments from people who noted the \"Americanisms\" in the examination and how challenging that was. I also wonder if there is room for future research related to how people do based on their English proficiency. -Response: Yes, this is really interesting and one that we frequently reflected on. Rwanda has gone through a significant transition of language skills and moving forward this issue would be significantly less of a problem as many of the newer residents aren’t francophone educated at secondary school. However, if the I-ITE was used in other settings, especially if a larger cohort, then this would make a really interesting piece of comparative research. We’ve expanded on this in the “future research work” section of the manuscript."
}
]
}
] | 1
|
https://f1000research.com/articles/9-1448
|
https://f1000research.com/articles/11-1105/v1
|
27 Sep 22
|
{
"type": "Case Report",
"title": "Case Report: Primary graft failure due to a reversed lenticule in Descemet Stripping Automated Endothelial Keratoplasty",
"authors": [
"Anahita Kate",
"Sayan Basu",
"Anahita Kate"
],
"abstract": "Introduction and importance: This report details the clinical features and management in a case of Descemet stripping automated endothelial keratoplasty (DSAEK) which had primary graft failure (PGF) due to an inverted yet attached lenticule. Presentation of case: A 66-year-old gentleman had poor visual recovery in the right eye after undergoing cataract surgery 12 years prior to presentation. The visual acuity was counting fingers and examination revealed endothelial decompensation. The patient underwent a DSAEK and postoperatively had a well attached lenticule. However, the cornea was edematous three weeks after the surgery and optical coherence tomography (OCT) revealed a reversed lenticule. The patient underwent a repeat DSAEK and had an uneventful postoperative course. The visual acuity was 20/40 after 7 months with a clear cornea and a well attached graft. Discussion: PGF is a rare complication following DSAEK which occurs due to poor endothelial function of the donor graft. Insertion of a reversed lenticule may get overlooked as a cause of PGF unless the graft edge profile is examined on an OCT scan. The graft in the current case was well attached despite its inverted position suggesting that graft adherence is perhaps not a function of the corneal endothelial pumps in isolation and may be driven by factors such as the intraocular pressure. Conclusion: A reversed DSAEK lenticule may have normal adherence to the host stroma and must be considered in cases with PGF. OCT of the graft edge is required for diagnosis before performing a repeat keratoplasty.",
"keywords": [
"Endothelial keratoplasty",
"Descemet stripping automated endothelial keratoplasty",
"inverted lenticule",
"graft adhesion",
"primary graft failure"
],
"content": "Introduction\n\nThe advent of endothelial keratoplasties has revolutionised the treatment of corneal endothelial disorders and has enabled the restoration of a near normal corneal anatomy and physiology.1,2 Also, the complications associated with a conventional penetrating keratoplasty (PK) such as suture related infections, vascularization and risks of an open sky procedure are drastically decreased with these lamellar surgeries.1,2 However, these procedures are not completely risk free and suboptimal results can ensue from graft detachment, rejection, chronic endothelial attrition, etc.1,3 Primary graft failure is one such complication that can occur following Descemet stripping automated endothelial keratoplasty (DSAEK) and is defined as corneal stromal edema which does not clear despite the presence of a well apposed graft.1,3 A donor graft with decreased endothelial count or loss of endothelial cells due to traumatic surgical manoeuvring are the most common causes of primary graft failure. This intraoperative manipulation of the graft is done primarily to ensure the correct orientation of the lenticule. Although several approaches exist to facilitate the same, it may still get attached in an inverted fashion, especially with very thin lenticules or in eyes with significant corneal scarring.4,5 Insertion of an inverted graft has not been reported as a cause of primary failure and so this report intends to describe the clinical features and management of primary graft failure in an eye with an attached yet reversed DSAEK lenticule.\n\n\nCase presentation\n\nA 66-year-old gentleman had complaints of intermittent pain and watering in the right eye after having undergone a small incision cataract surgery with a posterior chamber intraocular lens twelve years before presenting to us. This complaint had exaggerated in the three months prior to presentation and was now associated with a decrease in vision. There was no past history of any prior interventions, and the patient did not give any relevant family history. At presentation, his visual acuity in the right eye was counting fingers, while that in the left eye was 20/25. Slit lamp examination revealed an edematous cornea with epithelial bullae in the periphery (Figure 1). The anterior chamber was hazily seen and appeared normal with a stable intraocular lens. The posterior segment was hazily visible and revealed no abnormalities. The left eye had a visual acuity of 20/20 with a clear cornea and no guttate changes. A cataractous lens was present. Rest of the anterior and posterior segment examination was within normal limits. Specular microscopic examination revealed a healthy endothelial morphology and count in the left eye. A diagnosis of pseudophakic bullous keratopathy was made and the patient was planned for a DSAEK.\n\n(C, D) Image captured one month after the Descemet stripping automated endothelial keratoplasty (DSAEK) showing primary graft failure with a well attached graft. (E, F) Clear cornea with a attached DSAEK lenticule 2 weeks after the repeat endothelial keratoplasty.\n\nThe surgery was carried out under local anesthesia and followed a standard technique.6–8 Briefly, the central 9 mm of the host Descemet’s membrane was stripped. An 8mm trephination was carried out on a pre-cut donor tissue with an endothelial count of 2508 cells/mm2. The graft was inserted with a Sheets glide and apposed to the host stroma with a full chamber air tamponade of ten minutes. Postoperatively the visual acuity was counting fingers and the graft was edematous but well attached on the optical coherence tomography (OCT) scan (Figure 1). The patient was started on topical corticosteroids (prednisolone acetate 1%, six times/day) and antibiotics (moxifloxacin 0.5%, 4 times/day). The patient was reviewed again after three weeks and no improvement in either the visual acuity or the corneal clarity was noted. The OCT was repeated and the graft edge was included in the scan which showed a reversed orientation of the graft with the longer endothelial side adherent to the stroma (Figure 2). Furthermore, the irregular donor stromal surface facing the anterior chamber was also observed (Figure 2).\n\nThe patient underwent a repeat DSAEK, 6 weeks after the primary surgery. After the previous donor tissue was detached and removed, a new DSAEK lenticule, 7.5 mm in diameter, with an endothelial count of 2457 cells/mm2 was inserted. The rest of the surgical technique was similar to that of the first procedure. On the first postoperative day, the patient had a visual acuity of 20/60 with a well attached graft and a significant decrease in the corneal edema. The correct apposition and orientation of the graft was confirmed on OCT (Figure 2). The patient was continued on topical antibiotics and a tapering dose of topical corticosteroids. At the 7th month postoperative visit, the corrected visual acuity was 20/40 in the right eye. The cornea was clear with an attached lenticule, and the patient was maintained on a once daily dose of topical steroids (Figure 1). Figure 3 details the timeline of the patient from presentation to the last follow up.\n\nSICS: small incision cataract surgery, PCIOL: posterior chamber interocular lens, DSAEK: Descemet stripping automated endothelial keratoplasty, OCT: optical coherence tomography, VA: visual acuity.\n\n\nDiscussion\n\nSeveral studies have investigated the factors that affect graft attachment following a DSAEK.5,9,10 One of the key elements that facilitates good adherence of the lenticule is the endothelial pump action.9 A well-functioning endothelium also helps to clear the corneal edema. Hence primary graft failure and graft detachment is observed in eyes with a poor quality donor tissue or excessive intraoperative manipulation.1,3 Dislocation of the graft in such cases is probably mediated by the presence of fluid in the interface, which the dysfunctional endothelium is unable to overcome. And so, various modifications of the DSAEK technique have been employed which provide an additional means for graft apposition until the functional pumps can take over. These include surface massage to displace the interface fluid, use of venting incisions and roughening of peripheral stromal fibers to create stronger adhesions.5,9,10\n\nThe use of air tamponade and increase in intraocular pressure (IOP) has also been described for the same purpose. Vaddavalli et al and Bhogal et al independently studied the role of IOP in graft adherence.5,10 Both studies did not find a significant correlation between the two although Vaddavalli et al found an increasing rate of graft attachment with higher IOP. However, the experimental set up was devised to test the attachment under normal and high pressures and the role of hypotony was not assessed. Thus, IOP probably significantly contributes to good attachment of the donor lenticule as seen in the current case where a well attached graft was seen despite its inverted position. This also highlights the fact that there may be several other factors besides the endothelial pump action which bring about the binding of the two stromal surfaces. This needs to be taken into consideration especially in eyes which have a higher rate of graft dislocation such as aphakic eyes, vitrectomised eyes or eyes with glaucoma filtering surgery.1,3 Ensuring a watertight globe at the end of the surgical procedure will help prevent postoperative hypotony and decrease the risk of graft detachment.\n\nPrimary graft failure is a rare complication following DSAEK, with a reported rate of around 1%.3 The diagnosis of this entity is made in the presence of persistent corneal edema following a DSAEK. The normal time taken for the recovery of graft clarity following DSAEK ranges from three weeks to three months which can cause a delay in establishing the diagnosis of primary failure.3 As a result, the subsequent interventions required to visually rehabilitate the patient will also get stalled. However, a reversed lenticule is an exception to this as it can be detected in the early postoperative period and promptly managed. Several measures have been advocated to help ensure the insertion and attachment of the graft in its correct position. These include marking of the stromal surface, use of the double ring sign, intraoperative OCT, etc.4,11 However, the lenticule may get inverted despite these safeguards especially in cases with very thin grafts or in eyes with suboptimal clarity of the anterior chamber structures due to corneal scarring.\n\nAs highlighted by the current case, judicious use of an OCT in the weeks following the surgery can help identify the improper orientation of the lenticule in eyes with slow recession of corneal edema. Although documentation of an attached lenticule with an AS-OCT scan is a routine practice following endothelial keratoplasty, it is essential to include the peripheral graft area when capturing such images. An acute angle configuration of the graft edge has been described which indicates a stroma-to-stroma apposition with correct positioning of the endothelial layer.4 This additional information is vital as the mere presence of an attached lenticule does not signify its correct orientation.\n\n\nConclusion\n\nThe current report highlights a reversed DSAEK lenticule as a cause of primary graft failure. This clinical entity can be diagnosed early with the help of an AS-OCT in eyes with slow resolution of the corneal edema and a repeat intervention can be planned subsequently. Adhesion of the graft in DSAEK may depend on factors beyond functional endothelial pumps and an improved understanding of the same will help improve outcomes in eyes which are at a greater risk of having graft detachments.\n\n\nData availability\n\nAll data underlying the results are available as part of the article and no additional source data is required\n\n\nReporting guidelines\n\nFigshare. CARE guidelines. DOI: https://doi.org/10.6084/m9.figshare.20501754.12\n\n\nConsent\n\nWritten informed consent was obtained from the patient for publication of this case report and accompanying images.\n\n\nAuthor contributions\n\nSYB contributed to the study conceptualization, study investigation, methodology, manuscript editing and revision. AK contributed to the original draft preparation, study methodology and revisions.",
"appendix": "References\n\nAnshu A, Price MO, Tan DTH, et al.: Endothelial keratoplasty: a revolution in evolution. Surv Ophthalmol. 2012; 57: 236–252. PubMed Abstract | Publisher Full Text\n\nTerry MA: Endothelial keratoplasty: history, current state, and future directions. Cornea. 2006; 25: 873–878. Publisher Full Text\n\nSharma N, Maharana PK, Singhi S, et al.: Descemet stripping automated endothelial keratoplasty. Indian J. Ophthalmol. 2017; 65: 198–209. PubMed Abstract | Publisher Full Text\n\nTitiyal JS, Kaur M, Shaikh F, et al.: “Acute-angled bevel” sign to assess donor lenticule orientation in ultra-thin descemet stripping automated endothelial keratoplasty. BMJ Case Rep. 2019; 12: e227927. PubMed Abstract | Publisher Full Text\n\nBhogal MS, Angunawela RI, Bilotti E, et al.: Theoretical, experimental, and optical coherence tomography (OCT) studies of graft apposition and adhesion in Descemets stripping automated endothelial keratoplasty (DSAEK). Invest Ophthalmol Vis Sci. 2012; 53: 3839–3846. PubMed Abstract | Publisher Full Text\n\nChaurasia S, Vaddavalli PK, Ramappa M, et al.: Clinical profile of graft detachment and outcomes of rebubbling after Descemet stripping endothelial keratoplasty. Br J Ophthalmol. 2011; 95: 1509–1512. PubMed Abstract | Publisher Full Text\n\nGorovoy MS: Descemet-stripping automated endothelial keratoplasty. Cornea. 2006; 25: 886–889. Publisher Full Text\n\nMehta JS, Por Y-M, Beuerman RW, et al.: Glide insertion technique for donor cornea lenticule during Descemet’s stripping automated endothelial keratoplasty. J Cataract Refract Surg. 2007; 33: 1846–1850. PubMed Abstract | Publisher Full Text\n\nTerry MA, Hoar KL, Wall J, et al.: Histology of dislocations in endothelial keratoplasty (DSEK and DLEK): a laboratory-based, surgical solution to dislocation in 100 consecutive DSEK cases. Cornea. 2006; 25: 926–932. PubMed Abstract | Publisher Full Text\n\nVaddavalli PK, Diakonis VF, Canto AP, et al.: Factors affecting DSAEK graft lenticle adhesion: an in vitro experimental study. Cornea. 2014; 33: 551–554. PubMed Abstract | Publisher Full Text\n\nDelfazayebaher S, Feizi S, Javadi MA, et al.: Double-Ring Sign to Confirm Correct Orientation of Donor Lenticules During Descemet Stripping Automated Endothelial Keratoplasty. Cornea. 2015; 34: 980–984. PubMed Abstract | Publisher Full Text\n\nKate A, Basu S: SCARE Guideline Checklist-reversed DSAEK.docx. figshare. Online resource.2022. Publisher Full Text"
}
|
[
{
"id": "151994",
"date": "25 Oct 2022",
"name": "Ka Wai Kam",
"expertise": [
"Reviewer Expertise Cornea"
],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address 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 patient who underwent DSAEK and developed persistent corneal oedema after transplantation despite graft adherence.\n\nIn addition to the use of ASOCT, an attentive slit lamp examination with a narrow light beam and focusing on the graft edge may alert the clinician about a potential graft reversal. This may be highlighted as ASOCT may not be readily available in every eye centre.\n\nIn the report, the authors mentioned the use of Sheets glide in implanting the lenticule. Unlike in DMEK where some surgeons would use an S-stamp or an asymmetrical triangle mark at the graft periphery, graft orientation in DSAEK relies on a careful preparation and insertion process. It would be interesting to compare the different insertion techniques (Sheets glide, Busin glide, Endoglide) and look at the rate/risk of graft lenticule reversal - but in reality, there are multiple factors that could interfere with the final outcome.\nSimilarly, the authors may wish to elaborate on what in their opinion contributed to the graft reversal during the first transplantation, and what special precaution was taken during the second transplantation in order to avoid the same complication. This would be beneficial to novice EK surgeons in ensuring a correct graft insertion.\n\nIn places where grafts are scarce, reversal of a flipped endothelial keratoplasty may be attempted if the reversal had been noted earlier. In our experience, we had treated two flipped DSAEK buttons on postoperative day 1 by reversing the graft in the operating theatre. One of the two grafts cleared up after the reversal. This option may be considered especially in areas where corneal grafts are not as abundant.\n\nIs the background of the case’s history and progression described in sufficient detail? Yes\n\nAre enough details provided of any physical examination and diagnostic tests, treatment given and outcomes? Yes\n\nIs sufficient discussion included of the importance of the findings and their relevance to future understanding of disease processes, diagnosis or treatment? Yes\n\nIs the case presented with sufficient detail to be useful for other practitioners? Yes",
"responses": [
{
"c_id": "9185",
"date": "06 Jan 2023",
"name": "Sayan Basu",
"role": "Author Response",
"response": "1. In addition to the use of ASOCT, an attentive slit lamp examination with a narrow light beam and focusing on the graft edge may alert the clinician about a potential graft reversal. This may be highlighted as ASOCT may not be readily available in every eye centre. We thank the reviewer for their comment and agree that slit lamp examination can provide an understanding of the graft orientation especially in eyes with limited scarring and a good view of the anterior chamber. This point has been added to the discussion. 2. In the report, the authors mentioned the use of Sheets glide in implanting the lenticule. Unlike in DMEK where some surgeons would use an S-stamp or an asymmetrical triangle mark at the graft periphery, graft orientation in DSAEK relies on a careful preparation and insertion process. It would be interesting to compare the different insertion techniques (Sheets glide, Busin glide, Endoglide) and look at the rate/risk of graft lenticule reversal - but in reality, there are multiple factors that could interfere with the final outcome. We concur with the reviewer that understanding the rates of graft reversal in different insertion techniques will help identify the one associated with the least risk of graft reversal and this procedure can be adopted in eyes where in a higher risk of reversal is anticipated. This has been added to the discussion. 3. Similarly, the authors may wish to elaborate on what in their opinion contributed to the graft reversal during the first transplantation, and what special precaution was taken during the second transplantation in order to avoid the same complication. This would be beneficial to novice EK surgeons in ensuring a correct graft insertion. The first procedure was performed by a surgeon with relative inexperience and marking of the graft was not carried out. This could have resulted in the inversion of the graft during its insertion. This point along with measures to prevent the same has been added to the discussion to help surgeons early in the course of their training. 4. In places where grafts are scarce, reversal of a flipped endothelial keratoplasty may be attempted if the reversal had been noted earlier. In our experience, we had treated two flipped DSAEK buttons on postoperative day 1 by reversing the graft in the operating theatre. One of the two grafts cleared up after the reversal. This option may be considered especially in areas where corneal grafts are not as abundant. We agree that attempting a graft reversal is a viable option in low resource settings provided a reasonable time interval exists between the primary EK and the secondary procedure. This point has been added to the discussion."
}
]
},
{
"id": "151990",
"date": "01 Nov 2022",
"name": "Jaime David Martinez",
"expertise": [
"Reviewer Expertise Ophthalmology"
],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nSummary Interesting paper. This case presents a patient who had primary graft failure after a DSAEK graft. Graft failure was secondary to reverse graft tissue. The patient had a repeat DSAEK graft after surgery with the correct corneal graft position with a successful outcome. I like how they describe what to look for in these specific situations on OCT images to confirm the diagnosis. I think this is important as many cases are difficult to distinguish if the graft is in the correct position even with the reference maker.\nIt will be helpful for the surgeon to picture the different findings on OCT by doing the following:\nAdd in the Figure 2 legend the different types of angle configurations between cases. Maybe consider adding a geometrical line on the edges with acute and obtuse angles on OCT images.\n\nIs the background of the case’s history and progression described in sufficient detail? Yes\n\nAre enough details provided of any physical examination and diagnostic tests, treatment given and outcomes? Yes\n\nIs sufficient discussion included of the importance of the findings and their relevance to future understanding of disease processes, diagnosis or treatment? Yes\n\nIs the case presented with sufficient detail to be useful for other practitioners? Yes",
"responses": [
{
"c_id": "9186",
"date": "06 Jan 2023",
"name": "Sayan Basu",
"role": "Author Response",
"response": "We would like to thank the reviewer for reading and commenting on our submission. The required change has been made to the figure 2"
}
]
},
{
"id": "152987",
"date": "01 Nov 2022",
"name": "Denise Loya Garcia",
"expertise": [
"Reviewer Expertise Cornea",
"Corneal Transplant."
],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address 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 an interesting case of Primary graft failure due to reversed lenticule in Descemet Stripping Automated Endothelial Keratoplasty and a very valuable use of the anterior segment OCT.\nSlit-lamp biomicroscopy may not be as precise in edematous corneas as details may be easily missed, even by expert clinicians. Anterior Segment Tomography is a very useful tool in searching for clinical cues that may guide the clinician in the diagnosis. As clearly demonstrated by the authors in this case report, OCT of the graft edge may demonstrate the reversed position of the lenticule a detail that would be overlooked by biomicroscopy.\n\nIs the background of the case’s history and progression described in sufficient detail? Yes\n\nAre enough details provided of any physical examination and diagnostic tests, treatment given and outcomes? Yes\n\nIs sufficient discussion included of the importance of the findings and their relevance to future understanding of disease processes, diagnosis or treatment? Yes\n\nIs the case presented with sufficient detail to be useful for other practitioners? Yes",
"responses": [
{
"c_id": "9187",
"date": "06 Jan 2023",
"name": "Sayan Basu",
"role": "Author Response",
"response": "We thank the reviewer for their comments"
}
]
}
] | 1
|
https://f1000research.com/articles/11-1105
|
https://f1000research.com/articles/12-17/v1
|
06 Jan 23
|
{
"type": "Research Article",
"title": "3D-printing of porous structures for reproduction of a femoral bone",
"authors": [
"Giulia Alessandri",
"Gian Maria Santi",
"Paolo Martelli",
"Eleonora Guidotti",
"Alfredo Liverani",
"Gian Maria Santi",
"Paolo Martelli",
"Eleonora Guidotti",
"Alfredo Liverani"
],
"abstract": "Background: 3D-printing has shown potential in several medical advances because of its ability to create patient-specific surgical models and instruments. In fact, this technology makes it possible to acquire and study physical models that accurately reproduce patient-specific anatomy. The challenge is to apply 3D-printing to reproduce the porous structure of a bone tissue, consisting of compact bone, spongy bone and bone marrow. Methods: An interesting approach is presented here for reproducing the structure of a bone tissue of a femur by 3D-printing porous structure. Through the process of CT segmentation, the distribution of bone density was analysed. In 3D-printing, the bone density was compared with the density of infill. Results: The zone of compact bone, the zone of spongy bone and the zone of bone marrow can be recognized in the 3D printed model by a porous density additive manufacturing method. Conclusions: The application of 3D-printing to reproduce a porous structure, such as that of a bone, makes it possible to obtain physical anatomical models that likely represent the internal structure of a bone tissue. This process is low cost and easily reproduced.",
"keywords": [
"3D printing",
"porous strucutre",
"bone density",
"CT segmentation",
"bone model",
"bone tissue"
],
"content": "Introduction\n\nAging, rising obesity, and a lack of physical activity have all contributed to a significant increase in joint deterioration and bone abnormalities.1–3 The challenge is focused on making prototypes to replace or support bone parts. It is critical that the implant be functional and remain there without complications or performance deficits. Using traditional two-dimensional radiologic modalities, it is difficult to understand complicated defects, and the evaluation and classification of defects of various kinds are crucial steps to effectively manage clinical conditions. Making three-dimensional models provides both visual and tactile reproduction of bone anatomy, with the potential for better preoperative planning, thus helping to make complex interventions more precise and accurate.4 3D-printing and virtual surgery planning have found significant interest in the field of orthopaedics, leading to considerable advancement in preoperative surgical planning.5–7 Indeed, it is found that 3D models allow surgeons to visualize anatomy three-dimensionally and aid in the planning and execution of complex surgeries.8–10 3D printing of porous structure offers an attractive means to improve the fabrication of bone models and facilitate their understanding for both academic studies and surgical planning.11–13\n\nHere we present the process of 3D-printed porous structure of a femoral bone composed of different infill densities. Among the print parameters of the slicing software, it is possible to change the infill parameters and obtain a 3D print with differentiated densities, effectively replicating the appearance of a bone tissue: a compact and very dense outer part, a trabecular and less dense inner part, and the hollow marrow in the centre. The three-dimensional reconstruction of the anatomical section was performed following the well-established procedure in previous studies.14–16 In brief, by segmentation of medical images from CT scans, different internal zones of the bone are obtained, according to the intensity of the pixels in a grey scale. The study of the distribution of different infill densities as a 3D printing parameter was one of the key points of the research. In particular, the aim was to compare the infill density with the actual bone density obtained by reprocessing medical images. The possibility of obtaining a 3D-printed object that represents a bone both internally and from an external geometric point of view is the main goal of the research. This allows on the one hand a better understanding of the clinical case and on the other hand improves communication in the patient-doctor relationship.17 Moreover, the object studied allows students, professors or doctors to visualise and better understand bone tissue in their studies thanks to an object that faithfully simulates bone tissue. Biomaterials currently used for 3D printing in the medical and orthopaedic fields18,19 include polymers, materials widely used in additive manufacturing because of their ease of structural change due to relatively low melting points;20,21 metals and alloys, including titanium alloy, a compact, lightweight and highly corrosive-resistant material with osteointegrative properties that make it perfect for replacing missing parts or for support during alignment and surgical cutting.22,23 FDM is a manufacturing technology adapted for the fabrication of porous bone tissue at low cost, providing good mechanical properties.24–26 In fact, the 3D printing parameters are set by slicing software, which layers the imported 3D model. The formal accuracy is affected by the G-code setting.27–29 The affordability and capability of reliably reproducing a model show the value of 3D simulations in preoperative planning and implant trajectory prediction to prevent injury and accidental harm to adjacent bone components.30\n\nThe aim of this study is to obtain a low-cost model of a 3D-printed femoral bone with a porous structure that is formally equivalent to its real counterpart. with a view to the potential replacement of a diseased piece of bone tissue, the bone in question must be as suitable as possible for the patient, simulating the appearance and weight of the original bone by reproducing its internal density.\n\n\nMethods\n\nThe study describes a methodological process by which 3D-printed bone tissue can be obtained with a porous structure infill.\n\nMedical images of a right femur of a 30-year-old man were downloaded from an online database. The procedure involves 3D digital reconstruction of the medical images using 3D Slicer v4.11 software,31 which allows a reading based on the Hounsfield scale, indicating the level of radiation absorption by the bone based on its density32–35 (Figure 1).\n\nAccording to the Hounsfield scale, intervals are defined for each area of bone:\n\n• compact bone: 484 Hu to 1814 Hu;\n\n• spongy bone: 333 Hu to 484 Hu;\n\n• marrow: 230 Hu to 333 Hu.\n\nSelecting all the pixels in the region of interest (ROI) produces the three-dimensional anatomical model in which the three zones are placed inside each other. In order to select each of these areas separately, selection masks were defined (Figure 2). Each of this selection masks is identified with a distinct colour. This allows better visualization of the various parts of the 3D model that composed the bone. Once the different areas are identified, only those related to the bone are considered. They can then be exported to an .stl format file.\n\nTo automate the ROI selection process, a script in the Python language was developed that can reprocess a.jpg image of a CT and automatically identify varied selection masks.\n\nFigure 3 shows the flowchart of the script. By introducing an image packet from CT scan, the script converts and reads each image in grayscale, that is, in pixels in a colour scale of 0 to 256 shades of grey ranging from black to white, respectively. Image manipulation from code is possible through dedicated libraries such as OpenCV.36 The script analyses every single pixel in the images starting from the first pixel in the upper left corner and continuing to the right. Once the first row of an image is finished, it starts again by going down one row of pixels and so on. The script assigns each pixel one of 256 values depending on the gradation in the grayscale. Finally, it converts with a proportion the numbers from 0 to 256 into values from 0 to 100 by sending out a text with all the values. Defined three groups, values are assigned for each selection mask:\n\n• from 0 to 76 in the green mask (white pixel);\n\n• from 77 to 178 in the yellow selection mask (grey pixel);\n\n• from 179 to 255 in the brown selection mask (black pixel).\n\nBy processing the script, the image is outlined as shown in Figure 4. These three groups identify the three parts that make up the bone. Post-processing was required to exclude areas that are not of interest for selection.\n\nIn any case, this automated method needs to be further improved in terms of the pixel selection and categorization process. So, the methodology was carried forward with the manual reconstruction method.\n\n3D printing has been the technology used for making the physical anatomical model. However, a model preparation stage is required for additive technology. First, three meshes in .stl format were exported from 3D Slicer. In the digital environment of Blender v3.3.0,37 a series of simple Boolean operations were performed in order to obtain three distinct final models of compact bone, spongy bone and bone marrow, respectively. In order for the models to maintain their relative positions in space in the slicing environment, they were exported in the .3mf format.\n\nThe printer used for this study is a Fused Deposition Modeling (FDM) 3D printer, AnyCubic Predator. FDM technology involves the extrusion of thermoplastic materials using a heated nozzle that melts the material and deposits it, layer by layer, on a printing platform until the part is completed.38 PLA was chosen as the printing material because it is inexpensive, dense, versatile and easy to process.39 To obtain the diversity of the densities of the parts to be printed, a conversion comparing the density of bone with that of PLA filler was performed. Specifically, the density value of compact bone (equal to 2 g/cm3) and that of spongy bone (equal to 0.6 g/cm3)40 were compared with the density of PLA (equal to 1.3 g/cm3) (Figure 5).\n\nAssuming the maximum compact bone density, that is 100% infill, the density of spongy bone will be 46.2%, according to the following proportion:\n\nUltimaker Cura v5.0.041 is the slicing software used that allows you to manage printing parameters and export a G-code file. Among the various parameters, including layer height, printing temperature, printing speed, are those related to infill of the various layers. An infill density is set as a single, constant value throughout the object. By importing the three patterns in .3mf format to Ultimaker Cura, different densities can be set for each pattern. Gyroid infill was used since it appears to be the one most like the typical trabecular structure of bone. Applying the parameters shown in Table 1, a preview of the compact bone and the spongy bone is visualized (Figure 6). The 3D print for each zone of different density was made without external walls to achieve a more uniform 3D print object with visibly gradual infill.\n\n\nResults\n\nThe reconstruction and processing steps resulted in the three distinct three-dimensional parts of the areas of the bone. The areas of the obtained 3D digital models present smooth and compact surfaces in their entirety, maintaining a true-to-life appearance. Figure 7 shows the three obtained digital models of the bone and Figure 8 shows the final 3D printed models.\n\n\nDiscussion\n\nNowadays, polymeric materials do not reach a density equal to that of compact bone, but it is possible to study which among them can achieve the same mechanical and structural properties with the right printing settings. An interesting example is the use of PEEK or similar materials that can easily reproduce the structure of bone, but their use involves a complicated and expensive process.42–44 An interesting possibility is offered by bone tissue printing for the fabrication of fractured parts that should be removed or low in bone density through 3D Bioprinting.45,46 Another improvement concerns the use of an optimized infill for printing bone that is more accurate than the one used and more like a trabecular structure so that it is visually increasingly accurate and close to reality. For the future, it also desired to achieve the possibility of making a print that has not only three different zones but that the variation in density is continuous following the bone matrix and not differentiated by n zones.47 Another future development concerns the improvement of automation for the reconstruction phase to have an increasingly accurate as well as fast method.48\n\n\nConclusion\n\nBy focusing on patient specifics, 3D printing technology in orthopaedics can improve the understanding of clinical cases by creating patient-specific anatomical models. One interesting method to improve the creation of bone models and make them easier to understand for both academic research and surgical planning is variable density 3D printing. 3D printing porous structure allows to obtain an anatomical model that better represents its realistic counterpart in terms of shape, surface area, and weight. By three-dimensional reconstruction of a CT image of a femoral bone, interpreting zones of different densities, it is possible to obtain three zones corresponding to compact bone, spongy bone, and bone marrow, respectively. This process can be done in manual or semi-automatic mode, with a strong potential for automation. This involves writing an articulated computer language code to select and distinguish the different pixels constituting a CT image and categorize them automatically according to their intensity. Finally, through slicing software it is possible to customize the printing parameters, applying different infill densities per part, resulting in a 3D printed object with porous structure. The most interesting application of this process is precisely in making more lifelike bone structures, ensuring eventual comparable replacement. This study is a first approach to obtain a first 3D print model with porous structure. As a future development, it is important to achieve mechanical characteristics comparable to those of real bone tissue. It is first necessary to identify a material with suitable characteristics both structurally and biocompatibility for this application. Finally, it is good to optimize the internal infill geometry that ensures on the one hand the maintenance of mechanical characteristics and on the other hand a good surgical integration.",
"appendix": "Data availability\n\nMedical images used in this study can be downloaded from Embodi3d®, https://www.embodi3d.com/.\n\n\nReferences\n\nAmini AR, Laurencin CT, Nukavarapu SP: Bone tissue engineering: Recent advances and challenges. Crit. Rev. Biomed. Eng. 2012; 40(5): 363–408. PubMed Abstract | Publisher Full Text | Free Full Text\n\nNiu X, Li N, Du Z, et al.: Integrated gradient tissue-engineered osteochondral scaffolds: Challenges, current efforts and future perspectives. Bioact. Mater. 2023 Feb 1; 20: 574–597. PubMed Abstract | Publisher Full Text | Free Full Text\n\nPan RL, Martyniak K, Karimzadeh M, et al.: Systematic review on the application of 3D-bioprinting technology in orthoregeneration: current achievements and open challenges. J. Exp. Orthop. 2022 Dec 1; 9(1): 95. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMaryada VR, Mulpur P, Eachempati KK, et al.: Pre-operative planning and templating with 3-D printed models for complex primary and revision total hip arthroplasty. J. Orthop. 2022 Nov 1; 34: 240–245. PubMed Abstract | Publisher Full Text | Free Full Text\n\nLee AK-XX, Lin T-LL, Hsu C-JJ, et al.: Three-Dimensional Printing and Fracture Mapping in Pelvic and Acetabular Fractures: A Systematic Review and Meta-Analysis. J. Clin. Med. 2022 Sep 6 [cited 2022 Oct 26]; 11(18): 5258. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMiller JR, Singh GK, Woodard PK, et al.: 3D printing for preoperative planning and surgical simulation of ventricular assist device implantation in a failing systemic right ventricle. J. Cardiovasc. Comput. Tomogr. 2020 Nov 1; 14(6): e172–e174. PubMed Abstract | Publisher Full Text\n\nRezvani Ghomi E, Khosravi F, Neisiany RE, et al.: Future of additive manufacturing in healthcare. Curr. Opin. Biomed. Eng. 2021; 17: 100255. Publisher Full Text\n\nPark JW, Gang HG: Application of 3-dimensional printing implants for bone tumors. Clin. Exp. Pediatr. 2021 Oct 15; 65: 476–482. Publisher Full Text\n\nSun Z, Wee C: 3D Printed Models in Cardiovascular Disease: An Exciting Future to Deliver Personalized Medicine. Micromachines. 2022 Sep 22; 13(10): 1575. PubMed Abstract | Publisher Full Text | Free Full Text\n\nVenter RG, Kotze L, Ferreira N: A clinician-run 3D-printing laboratory for orthopaedic preoperative planning: an illustrative case series. South African Orthop. J. 2022 Sep 2; 61(3): 181–186. Publisher Full Text\n\nAlaña M, Lopez-Arancibia A, Ghouse S, et al.: Additively manufactured lattice structures with controlled transverse isotropy for orthopedic porous implants. Comput. Biol. Med. 2022 Nov 1; 150: 105761. 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Publisher Full Text Reference Source\n\nFrizziero L, Santi GM, Liverani A, et al.: Computer-aided surgical simulation for correcting complex limb deformities in children. Appl. Sci. 2020 Aug 1 [cited 2022 Jun 7]; 10(15): 5181. Publisher Full Text Reference Source\n\nFrizziero L, Trisolino G, Santi GM, et al.: Computer-Aided Surgical Simulation through Digital Dynamic 3D Skeletal Segments for Correcting Torsional Deformities of the Lower Limbs in Children with Cerebral Palsy. Appl. Sci. 2022 Aug 1; 12(15). Publisher Full Text\n\nKermavnar T, Shannon A, O’Sullivan KJ, et al.: Three-Dimensional Printing of Medical Devices Used Directly to Treat Patients: A Systematic Review. 3D Print Addit. Manuf. 2021 Dec 1; 8(6): 366–408. Publisher Full Text\n\nMirzaali MJ, Moosabeiki V, Rajaai SM, et al.: Additive Manufacturing of Biomaterials—Design Principles and Their Implementation. Materials (Basel). 2022 Aug 1; 15(15). PubMed Abstract | Publisher Full Text | Free Full Text\n\nBernardo MP, da Silva BCR , Hamouda AEI, et al.: PLA/Hydroxyapatite scaffolds exhibit in vitro immunological inertness and promote robust osteogenic differentiation of human mesenchymal stem cells without osteogenic stimuli. Sci. Rep. 2022 Feb 11 [cited 2022 Oct 23]; 12(1): 2333. PubMed Abstract | Publisher Full Text | Free Full Text Reference Source\n\nFatima N, Massaad E, Shankar GM, Shin JH: Structural Allograft versus Polyetheretherketone Implants in Patients Undergoing Spinal Fusion Surgery: A Systematic Review and Meta-Analysis. World Neurosurg. 2020 Apr 1 [cited 2022 Jul 22]; 136: 101–9. PubMed Abstract | Publisher Full Text\n\nHong G, Liu J, Cobos SF, et al.: Effective magnetic susceptibility of 3D-printed porous metal scaffolds. Magn. Reson. Med. 2022 Jun 1; 87(6): 2947–2956. PubMed Abstract | Publisher Full Text\n\nLam AD, Duffy GP: Early Tibial Component Fractures in a Cementless, 3D-Printed. Titanium Implant. Arthroplast Today. 2022 Dec 1; 18: 31–38. PubMed Abstract | Publisher Full Text | Free Full Text\n\nAlagoz AS, Hasirci V: 3D printing of polymeric tissue engineering scaffolds using open-source fused deposition modeling. Emergent Mater. 2020 Aug 1; 3(4): 429–439. Publisher Full Text\n\nRaeisdasteh Hokmabad V, Davaran S, Ramazani A, et al.: Design and fabrication of porous biodegradable scaffolds: a strategy for tissue engineering.2017 Nov 2 [cited 2022 Oct 23]; 28(16): 1797–825. Publisher Full Text\n\nFrizziero L, Santi GM, Liverani A, et al.: Paediatric orthopaedic surgery with 3D printing: Improvements and cost reduction. Symmetry (Basel). 2019 Oct 1 [cited 2022 Jun 7]; 11(10): 1317. Publisher Full Text Reference Source\n\nFerretti P, Santi GM, Leon-Cardenas C, et al.: Representative Volume Element (RVE) Analysis for Mechanical Characterization of Fused Deposition Modeled Components. Polymers (Basel). 2021 Oct 15 [cited 2022 Oct 30]; 13(20): 3555. PubMed Abstract | Publisher Full Text | Free Full Text Reference Source\n\nMele M, Campana G, Esculapio: Tecnologie additive: introduzione ai processi e alle strategie produttive.2021 [cited 2022 Oct 23]; Reference Source\n\nReddy MV, Eachempati K, Gurava Reddy AV, et al.: Error analysis: How precise is fused deposition modeling in fabrication of bone models in comparison to the parent bones? Indian J. Orthop. 2018 Mar 1; 52(2): 196–201. PubMed Abstract | Publisher Full Text\n\nZhang M, Lei M, Zhang J, et al.: Feasibility study of three-dimensional printing knee model using the ultra-low-dose CT scan for preoperative planning and simulated surgery. Insights Imaging. 2022 Dec 1; 13(1): 151. PubMed Abstract | Publisher Full Text | Free Full Text\n\nFedorov A, Beichel R, Kalpathy-Cramer J, et al.: 3D Slicer as an image computing platform for the Quantitative Imaging Network. Magn. Reson. Imaging. 2012 Nov 1 [cited 2022 Feb 14]; 30(9): 1323–1341. PubMed Abstract | Publisher Full Text | Free Full Text\n\nAmaliya R, Aisyah S, Hariyanto AP, et al.: Analysis study of doses distribution in lung cancer using 3D Slicer. J. Phys. Conf. Ser. 2021 Jul 1 [cited 2022 Apr 20]; 1943(1): 012047. Publisher Full Text\n\nBell D, Greenway K: Hounsfield Unit.2019 Oct 16 [cited 2022 Oct 25].Reference SourceReference Source\n\nNoda Y, Kaga T, Kawai N, et al.: Low-dose whole-body CT using deep learning image reconstruction: image quality and lesion detection. Br. J. Radiol. 2021 May 1 [cited 2022 Oct 27]; 94(1121): 20201329. PubMed Abstract | Publisher Full Text | Free Full Text\n\nYou Y, Niu Y, Sun F, et al.: Three-dimensional printing and 3D slicer powerful tools in understanding and treating neurosurgical diseases. Front Surg. 2022 Oct 14 [cited 2022 Nov 7]; 9. PubMed Abstract | Publisher Full Text | Free Full Text\n\nJoice AA, Rajkumar P, Deepa J, et al.: Colour discernment of tomatoes using machine vision system with OpenCV Python and Raspberry Pi. Indian J. Eng. Mater. Sci. 2022 Aug 1; 29(4): 502–508. Publisher Full Text\n\nblender.com: blender.org - Home of the Blender project - Free and Open 3D Creation Software. Blender.Com.2022.Reference Source\n\nQuodbach J, Bogdahn M, Breitkreutz J, et al.: Quality of FDM 3D Printed Medicines for Pediatrics: Considerations for Formulation Development, Filament Extrusion, Printing Process and Printer Design. Ther. Innov. Regul. Sci. 2022 Nov 1; 56(6): 910–928. PubMed Abstract | Publisher Full Text | Free Full Text\n\nRanakoti L, Gangil B, Mishra SK, et al.: Critical Review on Polylactic Acid: Properties, Structure, Processing, Biocomposites, and Nanocomposites. Materials (Basel). 2022 Jun 1; 15(12). PubMed Abstract | Publisher Full Text | Free Full Text\n\nCotterell B: Fracture and Life. Imperial College Press;2010.\n\nUltimaker Cura: un software di stampa 3D potente e facile da usare.[cited 2022 Dec 16].Reference Source\n\nGrivet-Brancot A, Boffito M, Ciardelli G: Use of Polyesters in Fused Deposition Modeling for Biomedical Applications. Macromol. Biosci. 2022 Oct 1; 22: 2200039. PubMed Abstract | Publisher Full Text\n\nMoby V, Dupagne L, Fouquet V, et al.: Mechanical Properties of Fused Deposition Modeling of Polyetheretherketone (PEEK) and Interest for Dental Restorations: A Systematic Review. Materials (Basel). 2022 Oct 1; 15(19). PubMed Abstract | Publisher Full Text | Free Full Text\n\nMohaghegh S, Hosseini SF, Rad MR, et al.: 3D Printed Composite Scaffolds in Bone Tissue Engineering: A Systematic Review. Curr. Stem Cell Res. Ther. 2021 Sep 6; 17(7): 648–709. Publisher Full Text\n\nGermaini MM, Belhabib S, Guessasma S, et al.: Additive manufacturing of biomaterials for bone tissue engineering – A critical review of the state of the art and new concepts. Prog. Mater. Sci. 2022 Oct 1; 130: 100963. Publisher Full Text\n\nImran R, Al Rashid A, Koç M: Review on computational modeling for the property, process, product and performance (PPPP) characteristics of additively manufactured porous magnesium implants. Bioprinting. 2022 Dec 1; 28: e00236. Publisher Full Text\n\nKanwar S, Vijayavenkataraman S: 3D printable bone-mimicking functionally gradient stochastic scaffolds for tissue engineering and bone implant applications. Mater. Des. 2022 Nov; 223: 111199. Publisher Full Text\n\nAl-Kharusi G, Dunne NJ, Little S, et al.: The Role of Machine Learning and Design of Experiments in the Advancement of Biomaterial and Tissue Engineering Research. Bioengineering. 2022 Oct 1; 9(10). PubMed Abstract | Publisher Full Text | Free Full Text\n\nDING-15:DING-15/TC_py: TC_py-v01 (v0.1).[Code] Zenodo.2022. Publisher Full Text"
}
|
[
{
"id": "228051",
"date": "29 May 2024",
"name": "Hannah S.",
"expertise": [
"Reviewer Expertise 3D printing for medical applications",
"orthopaedics",
"bone structure/porosity"
],
"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\nOverall, while the authors should be commended for their attempt at improving patient-specific 3D printed models, the work falls short in terms of any scientific rigor, especially compared to other similar research. Specific comments are provided below.\n- The manuscript can be improved from English language editing and some basic editorial corrections (e.g., \"FDM\" and \"PLA\" are not defined the first time it is used). - The introduction contains details that would be better suited for the methods section. - The automated process should not be included in the methods if a manual process was instead used. - For the manual segmentation process, it is not clear how the HU scale was determined or validated. - Using 100% infill to represent \"maximum compact bone density\" may not be accurate, as cortical bone has low (but not non-zero) porosity. - Saying the gyroid infill appears most like trabecular bone is unfounded. - Having three separate models with different infills seems like a weak solution considering the amount of existing literature detailing the design of graded porosity. - There is no validation of the model, mechanically or geometrically. This is a large gap in the study, since there is no way to say whether this is a useful model. - The Discussion is much too shallow and does not address limitations or consider other, more robust analyses of bone models or 3D printable bone-mimicking materials. - The conclusion includes remarks that are unsupported, since there was no validation of how well the printed model reflects actual anatomy.\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? No",
"responses": []
},
{
"id": "273191",
"date": "12 Jun 2024",
"name": "Marie-Luise Wille",
"expertise": [
"Reviewer Expertise CT imaging",
"3D modelling",
"Bone Tissue Engineering",
"3D Printing"
],
"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 approach to replicate a femoral bone via 3D printing and present a visually appealing anatomical model. However, in its current form the manuscript does not fulfill the requirements for indexing. It is unclear which aim the authors want to achieve with their research. It is stated multiple times that the ultimate aim of this research is to potentially replace the diseased bone, by having a structural equivalent 3D printed bone. This claim is unsupported and not valid, as current achievements in bone tissue engineering need to consider multiple aspects such as geometry, density, mechanical stability, vascularisation, cell proliferation, etc. which are not given in the current study. Further the use of PLA is very questionable for bone tissue engineering and it should be emphasised that PLA acts as a surrogate material. Further the claim that \"this study is a first approach to obtain a first 3D print model with porous structure\" is incorrect as there have been multiple previous studies (1-3) in the same area. Please see below specific comments: Introduction: - The second paragraph belongs to the method section. - Stronger emphasis should be given to the aim of the current research study, i.e. providing a visually bone mimicking anatomical model. The section about different biomaterials/metal alloys in 3D printing to replace missing parts is misleading. - The accuracy of the 3D printing model is only partially affected by the G-code setting. - what do the authors mean by 'formally equivalent' to its real counterpart? This needs to be reworded, as the PLA bone model is by no means equivalent to real bone.\nMethods: -Further details about the medical image data should be provided, i.e. scan resolution. - it is unclear how the HU are converted into the grayscale unit values of the range 0 to 256. - the python script the that is included in the manuscript indicates ranges of 30, 70, and 100 which are different to the 0, 76, 178 mentioned in the manuscript. - add manufacturer details for AnyCubic Printer. - please add details which PLA is used. PLA density varies between manufacturer and colourings. - while the spongy bone component was matched the cortical bone density is significantly lower in PLA. Why was not the overall density scaled? -it would be nice if the 3D printed model is CT scanned as well to compare the 'apparent densities'\n\nDiscussion - the discussion is very short and does not discuss the results of the current research at all. Mechanical properties are mentioned, but no mechanical testing has been performed.\n\nConclusion - the conclusion should be more realistic and reflecting on the current study. For example it is not the first approach to obtain a 3D print model with porous structures. There are other studies available (Reference 1-3, just to name a few are Clifton W, et al.(2019) [Ref-1]; Nägl K, et al. (2022) [Ref-2]; Bücking TM, et al. (2017) [Ref-3] ). - 'lifelike bone structures, ensuring eventual comparable replacement'... it is still a very long way to go for bone replacement and the current study cannot be claimed as a starting point. - this study should solely be considered as a way to produce low cost anatomical models (which justifies the use of PLA), but for any claims beyond this, this study lacks scientific proof (in vitro study (biocompatibility, cell proliferation, use of PLA instead of other materials...), mechanical testing, comparison to actual bone, etc.\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Partly\n\nAre sufficient details of methods and analysis provided to allow replication by others? Partly\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\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": []
}
] | 1
|
https://f1000research.com/articles/12-17
|
https://f1000research.com/articles/10-473/v1
|
15 Jun 21
|
{
"type": "Study Protocol",
"title": "Pre-clinical undergraduate students’ perspectives on the adoption of virtual and augmented reality to their dental learning experience: A one-group pre- and post-test design protocol",
"authors": [
"Kelvin I. Afrashtehfar",
"Jing-Wen Yang",
"A. Al-Sammarraie",
"Hui Chen",
"Musab H. Saeed",
"Jing-Wen Yang",
"Hui Chen",
"Musab H. Saeed"
],
"abstract": "Background: We live in a time where traditional education has rapidly incorporated online modalities due to the recent SARS-CoV-2 (COVID-19) safety measures such as social distancing. Regardless of these challenges, health education constantly strives to implement the best technologies available for an effective student deep learning outcome. Virtual (VR) and augmented reality (AR) in the dental pre-clinical stage may help stimulate students to better understand the foundation material prescribed in the curriculum. Most visual material available for students is still mainly based on 2D graphics. Thus, this study will attempt to evaluate the students' perceptions about implementing VR/AR technologies in the learning setting. Methods: A single-group pretest-posttest design will be implemented where students will be exposed to VR/AR and fill out two questionnaires, one before and one after the exposure. Conclusions: This project is intended to start once the institutional ethical approval is obtained. It is expected that the analysis from the current project will provide recommendations to improve the students' academic curriculum pre-clinical experience. The recommendations will be provided in the form of at least three scientific publications, with one publication for each subject area intended to be evaluated (i.e., head and neck anatomy, dental anatomy, and removable prosthodontics).",
"keywords": [
"Augmented Reality",
"Curriculum",
"Dental Students",
"Health Education",
"Immersion",
"Learning",
"Perception",
"Virtual Reality."
],
"content": "Introduction\n\nDigital technology has been adopted rapidly in student education. Virtual reality (VR) and augmented reality (AR) tools seem to result in an intense immersion1. The spatial perception about 3D objects may reproduce successful representation in a VR environment. However, to date, dental education is primarily aided by 2D image sources. It is believed that a VR teaching environment may deepen the understanding of dental anatomy2. In fact, a teaching environment with spatial representations improves the outcomes compared with 2D graphics, by keeping students motivated and providing them with an improved understanding3,4.\n\nThe study will be conducted on United Arab Emirates (UAE) pre-clinical dental students. It is essential to evaluate the potential for adopting a VR teaching environment for UAE pre-clinical dental students when understanding and learning about each tooth's characteristics, head and neck components, as well as partial and complete removable dental prostheses. It is equally important to assess the impact of VR in these courses, as perceived by the students. Thus, we hypothesized that students who learned using the traditional 2D teaching pre-clinical content (dental anatomy, head and neck, and removable prosthodontics courses) would not perceive a significant advantage in learning while implementing a VR/AR teaching environment.\n\nThis study aims to evaluate the dental students’ perceptions of the current undergraduate dental anatomy, head and neck anatomy, and removable prosthodontics training curriculum, as well as their perspectives on the incorporation of virtual learning into the curriculum.\n\nThe research questions that the project intends to answer are as follows:\n\nWhat are the dental students’ perceptions on the current undergraduate dental anatomy training?\n\n○ What are the dental students’ perceptions of the undergraduate dental anatomy training teaching during the 2020 summer session (fully online version) versus the 2021 fall session (blended learning)?\n\nWhat are the dental students’ perspectives on incorporating virtual learning into the dental anatomy training curriculum?\n\n○ What are the dental students’ perspectives of the incorporation of virtual learning into the dental anatomy training curriculum after experiencing the test?\n\nWhat are the dental students’ perceptions on the current undergraduate removable prosthodontics training curriculum?\n\n○ What are the dental students’ perspectives of the incorporation of virtual learning into the removable prosthodontics training curriculum after experiencing the test?\n\n\nProtocol\n\nDuring the 2021–2022 academic year, we will conduct a study with undergraduate students who have previously taken the evaluated courses.\n\nThe eligible students (chosen based on the following selection criteria) will be recruited via email and will obtain their consent documents via email. Electronic signatures will be required for indicating consent to participate. The participants will be sent the link to the electronic surveys (pre- and post-test self-administered questionnaires) via email. Copies of the informed consent form and research instruments are available in Extended data5.\n\nInclusion criteria. Ajman University second-year undergraduate dental students who took the dental anatomy, head and neck anatomy, and removable prosthodontics courses will be eligible to participate.\n\nExclusion criteria. Students who have not taken the dental anatomy course in the last academic year will not be eligible to participate.\n\nGiven the nature of the pretest-posttest design of this study, the prospective data will be collected at two moments6.\n\nThe first step consists of an online (host site TBD) questionnaire (see research instrument A in Extended data5) to obtain students' perceptions regarding the traditional curriculum of dental anatomy, head and neck, and removable prosthodontics courses, and to determine students’ potential acceptance of virtual learning.\n\nThe VR dental learning environment (i.e., use of VR glasses running the free-trial software [Head & Neck Anatomy version 3.0, 3D Tooth Atlas version 9.0, Complete Dentures version 1.0, and Removable Partial Dentures version 1.0; eHuman Inc., Fremont, Calif., USA] from their own mobile phones) will be available to the pre-clinical students for 30 minutes each, to test the student curriculum's appropriateness.\n\nAfter exposure to the VR/AR learning environment, the participants will provide feedback (see research instrument B in Extended data5). The post-test questionnaire will consist of selecting an answer for each prompt addressing their VR/AR experience and comparing it to their previous teaching methods when the courses were taken. Most answers will be categorical.\n\nThe results will be compiled and analyzed statistically to be presented as figures and tables.\n\nStatistical analysis. A statistical software (IBM SPSS Statistics, Version 27.0., IBM Corp.) will be used to perform all the statistical analyses. The variables will be class, gender, age, nationality, and type of high school. The demographic characteristics will be presented in tables.\n\nThe frequency of answers for each survey question that uses the \"Likert scale\" will be assessed with an independent t-test. The data from each of the examined curricula will be represented as mean and standard deviations. Non-parametric tests, such as the Kruskal-Wallis test and the Mann-Whitney U test, might be used for comparing ordinal variables, domains, and items between courses and academic years. In the case of multiple comparisons between academic years, the Bonferroni correction might be applied. In the case of multiple comparisons between the teaching courses in different academic years, the Bonferroni correction could also be applied.\n\nSample size calculation. In terms of the sample size, 132 or more measurements/surveys are needed to have a confidence level of 95% that the real value is within ±5% of the measured/surveyed value since one batch population size is approximately 200.\n\nThe security and confidentiality of the participants’ identities and electronic data files will be protected, and we will keep the data in encrypted files on a password-protected laptop computer. Electronic data will also be kept, for backup purposes, on a password-protected and encrypted external hard drive, and all non-electronic data will be stored in the locked office of a researcher (KIA or MHS).\n\nAll printed material will also be stored in a locked cabinet. Since all data will be de-identified, this will be publicly available indefinitely in a secure cloud-based repository such as Mendeley Data.\n\nThe researchers (KIA and MHS) will describe and provide recommendations based on the current pre-clinical curriculum for accepting or rejecting the consideration of virtual learning as a learning tool in the pre-clinical setting.\n\nIt is expected that the analysis from the current project will provide recommendations to improve the students' academic curriculum pre-clinical experience in the form of three publications in peer-reviewed Scopus-indexed journals7,8. This will be one publication for each subject intended to be evaluated (i.e., head and neck anatomy, dental anatomy, and removable prosthodontics). Additionally, we aim to produce at least one publication at a conference proceeding. This process may take less than 12 months from the moment of the ethical approval (Table 1).\n\nThe implementation activities will include giving support to other dental institutions interested in including VR/AR resources in their curricula, such as The University of Hong Kong and The Peking University.\n\nThe project involves humans considered a vulnerable population (i.e., undergraduate students) who are being exposed to a sensitive question, that is, to report their nationality. Nevertheless, students will have the option not to answer any of the questions or leave the study at any point. Moreover, this is considered to be a minimal risk study. Lastly, the nature of this study proposal requires Institution Research Ethics Review Board (IREB) approval before being conducted. The IREB application will be submitted in the summer of 2021.\n\nThis research project is intended to start once the IREB approval is obtained (fall 2021).\n\n\nDiscussion\n\nThe rapid incorporation of online (distance) learning modalities to traditional (face-to-face) education has become more visible in the last years. Due to SARS-CoV-2 (COVID-19) pandemic, the recent mandatory safety measures, such as social distancing, have abruptly extended the online learning modalities to most academic institutions. Apart from blended learning, health education has constantly strived to implement the most sophisticated technologies available for an effective student deep learning outcome. The current project adds an innovative perspective to the advancement of health education since 1) most visual material available for students is still mainly based on 2D graphics, and 2) implementing VR/AR technologies in the learning setting has never been more feasible.\n\nBy introducing VR/AR to the dental pre-clinical traditional, blended, and fully online learning contexts, we hope to stimulate students to better understand the foundation material prescribed in the dental undregraduate curriculum.\n\nOne of the study's strengths is that, most likely, we will meet the sample requirement according to the power calculation described in the data analysis section. Thus, it is expected to include a representative Middle Eastern geographically rich sample (i.e., primarily Iraq and Syria) and transfer our findings to other Middle Eastern settings.\n\nRegarding the proposal's limitations, the questionnaires to be used to assess students’ perspectives will not be validated. However, there is an important overlap with similar previous studies2–4.\n\nThis study could inform Asian dental educators of the feasibility of implementing AR/VR technologies to determine the effectiveness in a pre-clinical curriculum before expanding its use.\n\nThis project has been designed and originally planned to be conducted in the UAE (Dubai and Ajman). However, this project can also be replicated in China (Beijing and Hong Kong) as the coauthors have recognized the value of such an educational project and, in fact, they have submitted their respective IREBs.\n\n\nData availability\n\nNo underlying data are associated with this article.\n\nMendeley Data: Undergraduate students responses to VR/AR in their dental education. http://dx.doi.org/10.17632/2kycm5wwgt.15.\n\nThis project contains the following extended data:\n\n- Informed Consent Form - Extended.pdf (consent form).\n\n- A pre-test questionnaire-sample.pdf (research instrument A).\n\n- A post-test questionnaire-sample.pdf (research instrument B).\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).",
"appendix": "Acknowledgements\n\nKelvin Ian Afrashtehfar is an Assistant Professor in Prosthodontics at the College of Dentistry, Ajman University, UAE, an Associate Researcher in the Department of Reconstructive Dentistry and Gerodontology at the University of Bern School of Dental Medicine, Switzerland, and the Director of the Evidence-based Practice Unit in Ajman University.\n\nWe would like to thank the faculty and staff at the Ajman University College of Dentistry (M. Jaber, A. Jaghsi, R. Osman, Y. El-Karimi, and S. Ahmed) for agreeing to support one of the three sections of this research project.\n\nThe authors (KIA, AAHA, and MHS) thank Ajman University for supporting the present protocol study.\n\n\nReferences\n\nJoda T, Gallucci GO, Wismeijer D, et al.: Augmented and virtual reality in dental medicine: a systematic review. Comput Biol Med. 2019; 108: 93–100. PubMed Abstract | Publisher Full Text\n\nLiebermann A, Erdelt K: Virtual education: Dental morphologies in a virtual teaching environment. J Dent Educ. 2020; 84(10): 1143–1150. PubMed Abstract | Publisher Full Text\n\nMorales-Vadillo R, Guevara-Canales JO, Flores-Luján VC, et al.: Use of virtual reality as a learning environment in dentistry. Gen Dent. 2019; 67(4): 21–27. PubMed Abstract\n\nde Boer IR, Wesselink PR, Vervoorn JM, et al.: Student performance and appreciation using 3D vs. 2D vision in a virtual learning environment. Eur J Dent Educ. 2016; 20(3): 142–147. PubMed Abstract | Publisher Full Text\n\nAfrashtehfar KI, Al-Sammarraie AAH, Saeed MH: Undergraduate students responses to Virtual and Augmented Reality (VR/AR) in their dental education. Mendeley Data, V1, 2021. http://www.doi.org/10.17632/2kycm5wwgt.1\n\nGhanem H, Afrashtehfar KI, Abi-Nader S, et al.: Impact of a \"TED-Style\" presentation on potential patients' willingness to accept dental implant therapy: a one-group, pre-test post-test study. J Adv Prosthodont. 2015; 7(6): 437–445. PubMed Abstract | Publisher Full Text | Free Full Text\n\nAfrashtehfar KI, Eimar H, Yassine R, et al.: Evidence-based dentistry for planning restorative treatments: barriers and potential solutions. Eur J Dent Educ. 2017; 21(4): e7–e18. PubMed Abstract | Publisher Full Text\n\nAfrashtehfar KI, Bryant SR: Understanding the lived experiences of North American dental patients with a single-tooth implant in the upper front region of the mouth treated in a university setting by postgraduate dental students: A qualitative study protocol. JMIR Res Protoc. 2021. PubMed Abstract | Publisher Full Text"
}
|
[
{
"id": "137499",
"date": "22 Jun 2022",
"name": "Anand Marya",
"expertise": [
"Reviewer Expertise Orthodontics",
"Oral Health",
"Dental 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\nFirst of all I would like to thank the editor for inviting me to review this paper and secondly I would like to complement the authors on their hard work in compiling this manuscript. I do have some suggestions to help improve this manuscript:\n\nPlease do mention the mode of sampling which I think in this case is convenience sampling.\n\nIs the post-test questionnaire blinded? Bias elimination as it is a single-center study.\n\nWhat software/method has been used for sample size calculation and please expand on the power of the sample.\n\nPlease add notes on limitations of Likert scale.\n\nPlease consider the following papers: a. Jaber M, Al-Samarrai B, Al-Obaidee A, Varma SR, Karobari MI, Marya A. Does General and Specific Traits of Personality Predict Students’ Academic Performance?. BioMed Research International. 2022 Jan 4;2022. This is an insightful study into the academic performance of students based on their traits which are very important factors to consider for any educational perspective study.\nb. Veeraiyan DN, Varghese SS, Rajasekar A, Karobari MI, Thangavelu L, Marya A, Messina P, Scardina GA. Comparison of Interactive Teaching in Online and Offline Platforms among Dental Undergraduates. International Journal of Environmental Research and Public Health. 2022 Mar 8;19(6):3170. This an undergraduate level study where an analysis is done on the comparison between interactive teaching using various platforms.\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": "9089",
"date": "05 Jan 2023",
"name": "Kelvin Afrashtehfar",
"role": "Author Response",
"response": "The reviewer is acknowledged for providing suggestions for improving this manuscript. The authors have enjoyed reading and addressing every comment of the reviewer’s report. 1. 'Convenience sampling' was added. 2. The post-test questionnaire will be answered after the exposure using the same form used for the pre-test questionnaire. This has been added, too. 3. G*Power software (ver. 3.1.9.7; Heinrich-Heine-Universität Düsseldorf, Düsseldorf, Germany) will be used for sample size determination and power analysis. This has been included and cited. Reference: Kang H. Sample size determination and power analysis using the G*Power software. J Educ Eval Health Prof. 2021;18:17. doi: 10.3352/jeehp.2021.18.17. 4. To comply with the reviewer's comment, \"The development of the self-report survey tool includes an effort in maximizing validity and controlling for response biases (e.g., social desirability and acquiescent) in Likert scales,\" t was added. Reference: Kreitchmann RS, Abad FJ, Ponsoda V, Nieto MD, Morillo D. Controlling for Response Biases in Self-Report Scales: Forced-Choice vs. Psychometric Modeling of Likert Items. Front Psychol. 2019 Oct 15;10:2309. doi: 10.3389/fpsyg.2019.02309. 5. The suggested references Jaber et al. 2022 and Veeraiyan et al. 2022 were revised as indicated. The listed authors decided by consensus that the following references are pertinent to comply with the reviewer's suggestions. References: Veeraiyan DN, Varghese SS, Rajasekar A, Karobari MI, Thangavelu L, Marya A, Messina P, Scardina GA. Comparison of Interactive Teaching in Online and Offline Platforms among Dental Undergraduates. International Journal of Environmental Research and Public Health. 2022 Mar 8;19(6):3170. Lin GSS, Tan WW, Afrashtehfar KI. Exploring the Learning Experience of High-Performing Preclinical Undergraduate Dental Students: A Qualitative Study. Education Sciences. 2022; 12(11):801. https://doi.org/10.3390/educsci12110801 The authors appreciate the reviewer's intellectual effort and personal time to provide recommendations to benefit the appraised manuscript and the educational research community."
}
]
},
{
"id": "153097",
"date": "19 Oct 2022",
"name": "Kenneth Y. T. Lim",
"expertise": [
"Reviewer Expertise the learning sciences",
"pedagogy / andragogy",
"the use of VR / AR in learning"
],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nI am grateful for the opportunity to review your manuscript, which describes the perspectives of pre-clinical students with respect to the use of VR / AR within their curriculum. The plan for your study is coherently presented to your readers. You may wish to perhaps consider a more explicit review of the extant literature, so that the research questions may be justified in a more contextualised manner.\nYou may also wish to include a brief section on any anticipated risks, and any consequent (tentative) measures you anticipate you might be able to take, should the need arise to mitigate risks.\nIs the rationale for, and objectives of, the study clearly described?\nThe objectives are explicitly stated.\nThe rationale might be further improved by situating it more clearly within the literature. the authors are invited to elaborate on their understanding (as derived from literature) of the possible connections between the introduction of a spatial component in the dentistry curriculum through VR / AR and any resulting improvement in learner skillset.\n\nIs the study design appropriate for the research question?\nThe study design is appropriate for the research questions.\nAre sufficient details of the methods provided to allow replication by others?\nThe description of methods is currently not sufficiently specific. given that the study is yet to be conducted, an argument can reasonably be made that a more detailed description will follow upon completion of the actual study (which is pending IRB approval).\n\nAre the datasets clearly presented in a useable and accessible format?\nThe question of datasets is not relevant to this manuscript, as the manuscript represents a proposed study and not one which has already been completed.\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": "9090",
"date": "05 Jan 2023",
"name": "Kelvin Afrashtehfar",
"role": "Author Response",
"response": "Reviewer #2 is thanked for providing suggestions for improving this manuscript. The positive comments about the completeness of the study protocol are appreciated. 1.- It is already mentioned that \"this is considered a minimal risk study.\" However, it was added that students are considered a vulnerable population. Thus, the data collected does not reveal their identity, and anonymity is always preserved. Additionally, they are free to participate or withdraw from the study at anytime. There are no benefits or penalization to the student's grades whether they decide to participate or not. To avoid influencing the student's participation in response bias, an independent third party will collect the data, and any identifiable data will not be accessed until the course grades have been finalized. The informed consent will clearly state that participating in such a study has no educational benefit to the students. 2.- The rationale or justification of the appraised study is mentioned as \"It is essential to evaluate the potential for adopting a VR teaching environment for UAE pre-clinical dental students when understanding and learning about each tooth's characteristics, head and neck components, as well as partial and complete removable dental prostheses. It is equally important to assess the impact of VR in these courses, as perceived by the students.\" A hypothesis has also been provided. The implications \"Implications for practice and research\" agree and connect the introduction rationale with the discussion by stating, \"This study could inform Asian dental educators of the feasibility of implementing AR/VR technologies to determine the effectiveness in a pre-clinical curriculum before expanding its use.\" 3.- The author is thanked for providing a solution for addressing additional specifics regarding the methods. Thus, the reviewer's suggestion has been added to the manuscript text. \"More detailed description will follow upon completion of the actual study (IREB approved).\" The sentence \"This research project is intended to start once the IREB approval is obtained (fall 2021)\" was changed to \"This research project is intended to start in January 2023 as the IREB approval has been obtained.\" The authors acknowledge reviewer #2 for providing comments to improve the submitted manuscript. We are delighted with the result."
}
]
}
] | 1
|
https://f1000research.com/articles/10-473
|
https://f1000research.com/articles/10-1145/v1
|
11 Nov 21
|
{
"type": "Research Article",
"title": "Nutrient dynamics in water and soil under conventional rice cultivation in the Vietnamese Mekong Delta",
"authors": [
"Nguyen Vo Chau Ngan",
"Huynh Van Thao",
"Nguyen Dinh Giang Nam",
"Huynh Van Thao",
"Nguyen Dinh Giang Nam"
],
"abstract": "Background The evaluation of nutrient variability plays a crucial role in accessing soil potentials and practical intervention responses in rice production systems. Synthetic fertilizer applications and cultivation practices are considered key factors affecting nutrient dynamics and availability. Here, we assessed the nutrient dynamics in surface, subsurface water and soil under local water management and conventional rice cultivation practices in the Vietnamese Mekong Delta. Methods We implemented a field experiment (200 m 2) in the 2018 wet season and the 2019 dry season in a triple rice-cropping field. Eight samples of surface water, subsurface water (30–45 cm), and topsoil (0–20 cm) were collected and analysed during the rice-growing seasons. Results The results showed that N-NH 4+, P-PO 43- and total P peaks were achieved after fertilizing. Irrespective of seasons, the nutrient content in surface water was always greater than that of subsurface water (P<0.001), with the exception of N-NO 3-, which was insignificant (P>0.05). When comparing the wet and dry seasons, nutrient concentrations exhibited minor differences (P>0.05). Under conventional rice cultivation, the effects of synthetic fertilizer topdressing on the total N, soil organic matter (SOM), and total P were negligible in the soil. Higher rates of N fertilizer application did not significantly increase soil N-NH 4+, total N, yet larger P fertilizer amounts substantially enhanced soil total P (P<0.001). Conclusions Under conventional rice cultivation, N-NH 4+, P-PO 43- and total P losses mainly occur through runoff rather than leaching. While N-NO 3- loss is similar in surface water and subsurface water. Notably, nutrient content in soil was high; whilst SOM was seen to be low-to-medium between seasons. Future work should consider the nutrient balance and dynamic simulation in the lowland soil of the Vietnamese Mekong Delta’s paddy fields.",
"keywords": [
"nutrient availability",
"nutrient loss",
"surface water",
"subsurface water",
"soil",
"the Vietnamese Mekong Delta",
"water management"
],
"content": "Introduction\n\nThe Mekong Delta (MD) is the biggest rice-producing region in Vietnam (Clauss et al., 2018), accounting for approximately 55% of total national rice (Oryza sativa L.) outputs through intensive rice production systems (Uno et al., 2021). Here, double-and triple rice-cropping systems are the most commonly employed rice cultivation practices in the MD. Along with agronomic practices in intensive rice-based farming systems, a vast amount of fertilizer is typically applied to the paddy fields to obtain higher yields. For conventional rice cultivation in the MD’s paddy fields, the amount of fertilizer application in the wet season (WS) and dry season (DS) has been found to vary from 82–97 kg N ha−1, 22.64–22.69 kg P ha−1, and 29–32 kg K ha−1 (Stuart et al., 2018). Common rice practices typically use fertilizers, water, and seeds well exceeding recommended rates (Stuart et al., 2018, Connor et al., 2021). It is reported that the efficacy of N use by rice plants is generally relatively low, roughly 30–35%, while N loss to the environment is approximately 50% (Zhu and Chen, 2002). Moreover, Irfan et al. (2020) and Schröder et al. (2011) revealed that P utilization efficacy varies 10–15%, whereas P loss to the environment ranges 9.7–12.4% and 0.3–0.5% for surface runoff and subsurface leaching, respectively (Cho et al., 2011). In the rice field, fertilizer and water management regimes are key factors affecting transport, as well as the use efficacy of N and P (Qi et al., 2020). Yang et al. (2015) reported that different water and fertilizer management practices exported 13.1–31.7% N input to the environment, in which N loss through ammonia accounted for 69.6–83.5%. Furthermore, it has been noted that N loss from rice soils typically occurs through ammonia volatilization and nitrification-denitrification (Shankar et al., 2021), while loss of P was comparatively low due to the enrichment of Ca2+, Fe3+ and Al3+ oxides which can adsorb P in several mineral forms (Wang et al., 2015; Scalenghe et al., 2014). Several previous studies reported that nutrient migration was lost via surface water (SW) and sub-surface water (SbW) (Peng et al., 2011; Qi et al., 2020; Schröder et al., 2011; Wang and Huang, 2021). Thus, it has been suggested that higher amounts of fertilizer application under conventional rice cultivation and local water management regimes would largely result in increased nutrients in adjacent environments. To the best of our knowledge, quantitative variability of nutrients in SW, SbW and soil under conventional rice farming practices of the Vietnamese MD has not been previously studied. Therefore, this paper aims to explore the temporal-spatial dynamics of nutrients in the SW, SbW and soil of triple rice-cropping models both during the WS and DS under conventional local farming practices.\n\n\nMethods\n\nThis study was conducted at a local farmer's field in Long Tuyen district, Can Tho city, Vietnam (9°59’19”N, 105°36’14”E), from 2018 to 2019. The field experiment was located in a lowland soil, which applied triple-cropping rice, an intensive rice production system. According to Dong et al. (2012), the soil area was classified as Thionic Glycesol (International Union of Soil Sciences (IUSS) working group World Reference Base (WRB), 2015). The average weather data was annually recorded from 2015–2019 as follows: rainfall, 2,088.4 mm; humidity, 70.0–86.0%; sunshine, 2,467.4–2,695.4 hours (DONRE, 2020). The initial soil physicochemical properties were as follows: bulk density, 0.98 g cm−3; soil texture (sand, 1.9%, and clay, 66.4%); soil organic matter (SOM), 35.4 mg kg−1.\n\nThe size of the field experiment was 200 m2 (20 m × 10 m). The field was enclosed by a soil bank with plastic sheet coverage. The plastic sheet was buried 20 cm under the ground’s surface to secure against leaks or intrusion into the nearby fields. We conducted the field experiment in two seasons, including summer–autumn 2018 (wet season) and winter–spring 2019 (dry season). In summer–autumn 2018, the field experiment incorporated rice straw into the soil using a hand tractor. The straw was residue from the previous rice-growing season (spring–summer season 2018). The field witnessed a 10-day fallow period before sowing. In the winter–spring 2019 season, the field underwent a three-month natural flooding season. Before sowing, the field was drained and harrowed by a hand tractor. The rice crop calendar of the two field experiments is show in Table 1. Table 1 shows the rice farming practices during the wet season 2018 (summer–autumn) and dry season (winter–spring). The main practices comprise the schedule of soil preparation (ploughing), sowing, irrigation, fertilization, drainage, and harvest.\n\n† Date is formatted as dd/mm/yyyy; DAS = day after seeding.\n\nShort-duration rice varieties of OM4900 and OM6976 cultivars for the WS and the DS were used, respectively. The varieties were obtained from Cuu Long Delta Rice Research Institution (CLRRI), Vietnam. The maturity of the two rice varieties varied from 95 to 100 days. The selection of varieties was based on common use and edaphological adaptation in this region. Pre-germinated seeds were sown at 150 kg ha−1 under saturated soil by direct seeding. Water was supplied from a watershed near the field. Water management followed the locally typical water use practices. Water irrigation was started on the seventh day after seeding (DAS), re-irrigated 5–7 cm before fertilizing, always retaining a water level of 1–3 cm during heading and flowering, and openly drained ten days before harvesting. Multiple drainages, which are a simplified form of alternative wetting and drying (AWD) typically conducted in the VMD (Uno et al., 2021), were performed whenever water level naturally decreased 10 cm below the soil surface for the remaining cultivation period.\n\nWe applied synthetic fertilizers according to locally conventional rice cultivation. In the WS, 129.5 kg N ha−1 and 75 kg P2O5 ha−1 were used in total. These topdressings were applied at intervals of 10, 20, and 47 DAS. The fertilizers were applied as follows: 55/42.5/32 kg N ha−1, 25/25/25 kg P2O5 ha−1. In the DS, chemical fertilizers were used with a total amount of 90 kg N ha−1, 94 kg P2O5 ha−1, and 70.5 kg K2O ha−1. Fertilization was split into four intervals on days 10, 16, 26, and 47 DAS. The quantity of fertilizer was as follows: 30/15/30/34 kg N ha−1, 30/0/30/34 kg P2O5 ha−1, and 22.5/0/22.5/22.5 kg K2O ha−1. Nitrogen (N), phosphorous (P), and potassium (K) were applied based on the application of urea, superphosphate, and potassium chloride fertilizer. Applied fertilizer quantities for field experiments are shown in the Table 2.\n\n† Fertilizers applied were: N, P2O5, K2O (kg ha−1).\n\nTopsoil samples (10 cm) were collected by an auger with a 3.5 cm diameter. In the WS of 2018, we collected a soil sample before sowing to determine the soil's initial physicochemical properties. During the growth period, soil samples were taken on days 9, 13, 19, 27, 39, 53, 65, and 72 DAS. In the dry season of 2019, soil samples were collected on days 7, 14, 21, 29, 44, 52, 61, and 72 DAS. Samples were collected at five cross-sectional sites (four corners and one midpoint) and mixed to a similar weight to achieve a compromised sample. Fresh soil samples were removed of visible biomass, and air-dried and sieved at 2 mm. Soil texture was measured by sieving particle sizes to separate out coarse sand from the finer particles and the silt and clay contents were then determined by measuring the rate of settling of these two separates from the suspension in water according to the Robinson pipette method (Carter and Gregorich, 2008). Bulk density samples were collected by core samplers and the cores were dried in an oven at 110oC until the weight was constant in accordance with the Core method (Blake and Hartge, 1986). Soil organic matter (SOM) was oxidized by a K2Cr2O7-H2SO4 oxidation procedure and titrated using (NH4)2Fe(SO4)2(H2O)6 solution (Walkley and Black, 1934). NH4+ was extracted by KCl 1M (1:10 soil/extract (wt:vol)) and measured according to the indophenol blue colorimetric method (Lu, 2000). Total N (TKN) was digested in the digestion tablets (K2SO4, CuSO4, and Se) and H2SO4 solution at 375oC, then the digest was analyzed for NH4+ by the automated phenate method according to the Kjeldahl method (Bremner, 1996). Total P (TP) was digested in sulphuric acid-hydrogen peroxide-hydrofluoric acid (H2SO4-H2O2-HF) and detected by the molybdenum blue method (Bowman, 1988).\n\nWe also established a similar sampling program among SW, SbW, and soil. Likewise, SW samples were collected at soil sampling points and then mixed to obtain a joint representative sample. For SbW sampling, we installed five PVC pipes (120 cm in length and 9 cm in diameter) around the selected sampling points. The pipe was perforated by 2 mm holes and covered underneath by a lid. The perforated pipes were 15 cm in length. A plastic net of 2 mm was wrapped around the perforated pipe to avoid sediment intrusion. At each selected site, the pipe was anchored under the soil surface at 0.45 m depth. A lid was used to cover the pipe during non-sampling periods. NH4+, NO3−, PO43− and TP were analysed according to Standard Methods for the examination of Water and Wastewater (SMEWW) (APHA, 1998): NH4+ was detected by the phenate method (SMEWW 4500-NH3 F), NO3− was analysed by the automated hydrazine reduction method (SMEWW 4500-NO3− G), PO43− was determined by the ascorbic acid method (SMEWW 4500-P E), TP was measured by the persulfate method for simultaneous determination of total phosphorus (SMEWW 4500-P J).\n\nWe assessed the nutrient variation in SW, SbW, and soil between the WS and DS and compared the concentration of water environmental parameters between SW and SbW. The differences between levels of each factor were analysed assuming equal variances (Student’s t-test) at a significant level of P = 0.05 after passing the normality test (Shapiro-Wilk) (P > 0.05). All computations were performed using R stats Version 4.2.0 (R Project for Statistical Computing, RRID:SCR_001905).\n\n\nResults\n\nNutrient variations in SW and SbW are shown in Figure 1. The concentrations of N-NH4+, N-NO3−, P-PO43− and TP varied largely in the SW while remained relatively stable in the SbW. In particular, the nutrient values of the SW varied as follows: N-NH4+ (WS, 1.14–4.25 mg L−1; DS, 1.03–4.09 mg L−1), N-NO3− (WS, 0.46–1.03 mg L−1; DS, 0.27–0.97 mg L−1), P-PO43− (WS, 0.23–0.96 mg L−1; DS, 0.23–0.81 mg L−1), and TP (WS, 1.06–4.89 mg L−1; DS, 0.81–4.24 mg L−1), while the concentration of nutrients in the SbW varied as follows: N-NH4+ (WS, 0.34–0.73 mg L−1; DS, 0.24–0.71 mg L−1), N-NO3− (WS, 0.22–0.86 mg L−1; DS, 0.23–0.81 mg L−1), P-PO43− (WS, 0.03–0.08 mg L−1; DS, 0.02–0.07 mg L−1), and TP (WS, 0.51–1.19 mg L−1; DS, 0.41–1.08 mg L−1). The ratio of N-NH4+/N-NO3− in the SW was consistently higher than in the SbW. Particularly, the rate of N-NH4+/N-NO3− in the WS and DS varied by (SW, 1.4–6.9; SbW, 0.57–1.63) and (SW, 1.27–11.7; SbW, 0.72–2.0). It is noted that the concentration of N-NH4+, P-PO43− and TP in the SW was consistently higher than that in the SbW (P < 0.05), while the value of N-NO3− was insignificant between the SW and SbW (P > 0.05). As observed, the highest peaks of these parameters were reached after fertilizing. It is likely that the higher concentration of N-NH4+, P-PO43− and TP in the SW were observed during the fertilizing period. NO3− increased in the SbW during the fertilizing period, while the SW only rose in the WS and was more complex in the DS (Figure 1). Although the temporal-spatial dynamics of the SW and SbW parameters were complex over the rice-growing period, statistical analysis indicates that for N-NH4+, N-NO3−, P-PO43− and TP; no significant differences between the WS and DS were seen (Table 3).\n\nVertical dotted lines indicate the times of the synthetic fertilizer application. F1, F2, F3 and F4 depict topdressing of fertilizer 1, 2, 3, and 4, respectively.\n\nFigure 2 shows the N-NH4+, TN, SOM, and TP variation in the soil paddy field over the WS and DS. In the WS, the concentration of soil chemical properties varied as follows: N-NH4+ (21.5–38.0 mg kg−1), TN (2.37–2.90 g kg−1), SOM (37.6–48.5 g kg−1), and TP (0.65–1.69 g kg−1), while the DS fluctuated as follows: N-NH4+ (18.41–29.6 mg kg−1), TN (1.53–4.48 g kg−1), SOM (38.7–44.9 g kg−1), and TP (0.35–0.89 g kg−1). The concentration of N-NH4+ increased relatively after fertilizer application in both the WS and DS. Likely, a similar trend was seen in the TN during the DS. However, the effects of synthetic fertilizer topdressing on the TN (in the WS), SOM, and TP were neglectable. Statistically, N-NH4+, TN, and SOM slightly increased in the WS (P > 0.05), while TP significantly increased (P < 0.001) (Table 4).\n\nVertical dotted lines indicate the times of the synthetic fertilizer application. F1, F2, F3 and F4 depict topdressing of fertilizer 1, 2, 3, and 4, respectively.\n\nTP = total phosphorus; TN = total nitrogen.\n\n\nDiscussion\n\nOur study assessed nutrient variability in the SW and SbW through the WS and DS under typical water management and conventional rice practices in the Vietnamese MD. The study found that the concentration of N-NH4+, N-NO3−, P-PO43−, and total P in the SW exhibited a relatively large variation in the WS and DS (Figure 1). This could be partly attributed to shifting water levels (rainfall and irrigation) and fertilizer application (Qiao et al., 2012). The alteration of SW levels could likely increase/decrease the denseness of constituents regarding the concentration/dilution in the rice field. In this study, we did not record the water levels as the farmer let water flow free on the paddy field. Thus, the interdependence between water levels and nutrient dynamics remains uncertain. However, fertilizer topdressings could also potentially stimulate the dynamic mineralization processes within the rice paddy field. Here, we found that higher N fertilizer applications (39.5 kg N ha−1) in the WS slightly increased the average N-NH4+ and N-NO3− concentration by 5.83% and 13.8%. In contrast, higher P fertilizer utilization (15 kg P2O5 ha−1) in the DS was indistinguishable in cases of P-PO43−, and TP (Table 2). Thus, we suggest that an interaction between water levels and fertilizer application rates on the dynamics of N-NH4+, N-NO3−, P-PO43−, and TP in the Vietnamese MD’s paddy fields should be considered for further work.\n\nOur study showed that the N-NH4+, N-NO3−, P-PO43−, and TP contents in the SW were consistently higher than that of the SbW simultaneously (P < 0.001), irrespective of factors including fertilizer application rate and seasonal variation (Ngan et al., 2021). This means that nutrient loss mainly occurred through the SW. The lower nutrient concentrations in the SbW may also be partly explained by various transformation processes or plant uptake during percolation regression. For instance, the N-NH4+ could be reduced during leachate due to volatilization, nitrification (N-NH4+ to N-NO3−), and rice roots uptake (Hou et al., 2007). Furthermore, P-PO43− reduction could also be explained by rice plant uptake, binding onto soil minerals and being more prone to removal through surface runoff (McDowell et al., 2001). In line with our findings, Cho et al. (2011) found that leaching downward subsurface waters were responsible for 6.4–9.8% and 0.2–0.3% of N and P losses, respectively, while N and P losses via surface runoff accounted for 34.3–42.6% and 3.8–5.3%. It has been reported that nutrient loss during the rice-growing period is more relevant to the fertilizer application rate. Cui et al. (2020) confirmed that fertilizer applications significantly impacted N and P losses from surface runoff, with increased fertilizer application rates significantly increasing N loss through surface runoff. Besides, Qiao et al. (2012) showed that N loss via surface runoff and percolation positively correlated with fertilizer application rate. Also, the rate and timing of fertilizer application in the field influenced the N concentration loss over surface runoff (Li et al., 2018). These studies strongly supported our findings.\n\nOur study found that N-NO3− concentrations showed no significant difference between the SW and SbW, while N-NH4+ in the SbW was consistently lower than in the SW (Ngan et al., 2021). It is well-known that N-NH4+ in the rhizosphere area generally lowers the N-NO3− as rice prefers N-NH4+, up taking N-NH4+ faster than N-NO3−. Moreover, nitrification progression also occurs very fast in the rhizosphere (Li et al., 2015; Li et al., 2018). In the rice paddy field, N-NH4+ was simulated by both passive and active uptake, while N-NO3− was solely simulated by a passive uptake (Šimůnek and Hopmans, 2009). This meant that rice uptakes more N-NH4+ than N-NO3−. In line with these findings, Kirk and Kronzucker (2005) depicted that rice could absorb N-NH4+ from 60% to 85%, while 85% of N leaching loss exists in the N-NO3− form; a higher concentration of N-NO3− found below the soil surface (mostly in the rhizosphere) resulted in it being quickly transported to the soil surface (Mo’allim et al., 2018). As such, it is evident that N-NO3− tends to be high in SbW, which is consistent with our findings. However, it is noted that the change between N-NH4+ and N-NO3− pertains to rainfall, surface runoff, and irrigation (Li et al., 2015). Thus, we propose that the underlying nutrient dynamics in rice paddy fields under different water management regimes and differences of rice farming practices in the lowland soils of the Vietnamese MD should be further studied in future work.\n\nThis study described the soil chemical properties during the rice-growing period under conventional rice practices in the Vietnamese MD. Variability of N-NH4+, TN, SOM, and TP during rice growth was comparable to the previous studies undertaken in the lowland soils of the MD (Minamikawa et al., 2021; Vo et al., 2018; Uno et al., 2021). According to Hung et al. (2016) and Tanaka et al. (2014), soil properties in our study were characterized by medium-high TN, high N-NH4+, low-to-medium SOM, and high-to-very high TP. Thus, with respect to ensuring soil responsiveness to rice nutrient demand, reducing the N and P fertilizer application rate, and increasing SOM to a feasible degree should be considered in conventional rice practices in the Vietnamese MD.\n\nWe found that soil N-NH4+ and TN concentration slightly increased after fertilizing. Higher N-fertilizer application (39.5 kg N ha−1) in the WS insignificantly increased the N-NH4+ and TN in the soil in comparison to that of the DS (P > 0.05). However, higher fertilizer application of 15 kg P2O5 ha−1 significantly increased TP in the soil paddy field (P < 0.001). It is indicated that the fertilizer application moderately boosted the dynamic of N availability in soil. In agreement with our study, Dong et al. (2012) confirmed that available N slightly increased with chemical fertilizer application but significantly increased with organic matter additions. It is noted that the significant difference in TP could be likely due to the excessive P fertilizer application rates in the WS, while utilization efficacy and loss of P are usually low (Irfan et al., 2020; Schröder et al., 2011; Cho et al., 2011).\n\nSOM plays an inevitable role in promoting nutrient availability and improving soil fertility. Our study found that SOM change was minor during rice growth. This implied that the regression of organic matter mineralization/decomposition could occur slowly. Moreover, no organic matter was added to the soil in the paddy field. Thus, organic matter ineffectively contributed to nutrient availability in the soil. In the soil, change of SOM depends on temperature, pH, microbial growth, soil management, organic matter amendment, and C/N ratio (Tanaka et al., 2014).\n\n\nConclusions\n\nThis study examined the temporal-spatial variability of nutrients in SW, SbW, and soil of a paddy field in the WS and DS under typical water management and conventional cultivation techniques. We found that nutrient content in the SW showed a high fluctuation during the rice-growing period, while stability was observable in the SbW. After fertilizer application, the highest peaks of N-NH4+, P-PO43− and TP parameters in the SW and SbW were observed. The concentrations of N-NH4+, P-PO43-, and TP in the SW were all-time higher than that of the SbW. While N-NO3− concentration was insignificant between the SW and SbW. The seasonal nutrient variations were insignificant in both the SW and SbW. Our findings showed that soil properties were characterized by medium-high TN, high N-NH4+, low-to-medium SOM, and high-to-very high TP. Higher N fertilizer application slightly increased the N-NH4+ and TN dynamic, while TP significantly increased along with increasing P fertilizer application rate in the WS. SOM showed stability during both the WS and DS. We suggest that nutrient loss estimations and dynamic simulations in the lowland soil of the Vietnamese MD’s rice paddy fields should be considered for further work.\n\n\nData availability\n\nFigshare: Underlying data for ‘Nutrient dynamics in water and soil under conventional rice cultivation in the Vietnamese Mekong Delta’. ‘Paddy field in the Vietnamese Mekong Delta 2021’, https://doi.org/10.6084/m9.figshare.16499508.v1 (Ngan et al., 2021).\n\nThis project contains the following underlying data:\n\n• Supplementary file – 2021.xlsx\n\nData are available under the terms of the Creative Commons Zero “No rights reserved” data waiver (CC0 1.0 Public domain dedication).",
"appendix": "Acknowledgements\n\nWe thank Le Huu Thinh family who allowed us to process the experiments on their paddy rice field, Tran Thi Thuy Loan, Nguyen Thi Thu Thao, Pham Thi Huynh Nhu at Can Tho University for assisting the experiment activities, the E2 team (Can Tho University Improvement Project) for their administrative support to perform this study. Last but not least, thanks to Nigel Downes for proofreading the manuscript.\n\n\nReferences\n\nAPHA: Standard methods for the examination of water and wastewater. Washington DC, USA:American Public Health Association, American Water Works Association and Water Environmental Federation;20th ed.1998.\n\nBlake GR, Hartge KH: Bulk density and particle density. ‘Methods of soil analysis. Part 1’. Agronomy Monograph 9. Klute A, editor.Madison, WI:ASA and SSSA;1986; pp. 363–382.\n\nBowman RA: A rapid method to determine total phosphorus in soils. Soil Sci. Soc. Am. J. 1988; 52: 1301–1304. 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}
|
[
{
"id": "135271",
"date": "04 May 2022",
"name": "Koki Toyoda",
"expertise": [],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis manuscript reports valuable field data regarding nutrient dynamics in a paddy field. The results of differences between two depths and two seasons are interesting and worth to be published. However, there are several parts that are poorly explained or lack clearness. I commented directly on the manuscript. These comments should be properly amended.\nThe annotated manuscript can be found at the following link: https://f1000researchdata.s3.amazonaws.com/linked/419959.Koki_Toyota_review_ngan_nguyen-vo-chau.pdf\n\nIs the work 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": "9043",
"date": "15 May 2023",
"name": "Ngan Nguyen-Vo-Chau",
"role": "Author Response",
"response": "Response to reviewer: Thank you very much for giving us constructive comments that helped us improve this paper immensely. All your comments have been carefully read and revised to elucidate underlying aspects. Our responses to your comments are as follows: Abstract: 1.1 Comment 1 & comment 2: please describe the contents. I guess the following expression is preferable. \"Surface water, subsurface water, and topsoil samples were collected eight times during the ***\"; during the rice-growing seasons to estimate/clarify ????? Answer: Thank you so much for your commendation. The recommendation has been adopted. We revised it as follows “Surface water, subsurface water (30–45 cm), and topsoil (0–20 cm) were collected eight times during the rice-growing seasons to clarify its nutrient dynamic”. 1.2 Comment 2: describe the P-value in case of P>0.05. Answer: We rephrased the sentence as follows: “no significant difference was disclosed (P>0.05)” to make it clearer to readers. Detailed interpretation of the p-value was cleared in the methodology section “A P-value less than 0.05 was statistically significant, while a P-value greater than 0.05 indicated no effect. Significantly different comparison was considered at ***P < 0.001, **P < 0.01, *P < 0.05 and †P > 0.05.” 1.3 Comment 3: “total P losses mainly occur through runoff rather than leaching” à better to describe the results supporting this conclusion. Answer: We added the result to support the conclusion. It is as follows “Under conventional rice cultivation, the low concentration of N-NH4 +, P-PO4 3- and total P in the subsurface water indicated that nutrient losses mainly occur through runoff rather than leaching”. 1.4 comment 4: “While N-NO3- loss is similar in surface water and subsurface water” à not clear. How do the authors define \"loss\"? Answer: We agree with the reviewer that the conclusion is not clear and did not strongly support the conclusion. It is, therefore, eliminated. 1.5 Comment 5: “dynamic simulation” à not clear Answer: We revised the sentences as follows: “Future work should consider the nutrient balance and nutrient dynamic simulation in surface and subsurface of the lowland paddy soil in the Vietnamese Mekong Delta”. Introduction: 2.1 Comment 1: “common practice” à common practice in the MD or VN Answer: It has been corrected in the context of the mentioned paper. The revision is as follows: “Common practices in the MD typically use fertilizers, water, and seeds well exceeding recommended rates”. 2.2 Comment 2: “recommend rate” by ?? Answer: We added more information to make it clear to readers. The supplementary information is as follows: “Common practices in the MD typically use fertilizers, water, and seeds well exceeding recommended rates sustainable rice farming practices combined in the national program “One Must Do, Five Reductions” (1M5R)”. 2.3 Comment 3: “Nutrient migration was lost” à not clear Answer: We revised this sentence to make it clearer to readers which is as follows: Several previous studies reported that nutrient losses primarily occurred via surface water (SW) and sub-surface water (SbW). Methods: 3.1 Comment 1 “sand, 1.9%, and clay, 66.4%” à silt ??%. Answer: We supplemented the percentage of silt as follows: “soil texture (sand, 1.9%, silt, 31.7%, and clay, 66.4%)”. 3.2 Comment 2 “soil organic matter (SOM), 35.4 mg kg −1” à too low value Answer: Thank you so much for recognizing the low value. This was our miswriting when it came to filling the unit. It has been adopted and revised as follows: “soil organic matter (SOM), 35.4 g kg −1 “ Results: 4.1 Comment 1: “P > 0.05” à describe the P value Answer: Please see our response in “comment 1.2” 4.2 Comment 2: N-NH 4 + (18.41–29.6 mg kg −1) à 18.4 Answer: It has been adopted. Revision is as follows: “N-NH 4 + (18.4–29.6 mg kg −1)” Discussion: 4.1 Comment 1: “This means that nutrient loss mainly occurred through the SW” à This is a bit radical interpretation. Please explain more logically why the authors describe \"mainly\". In addition to this hypothesis, uptake of the nutrients by rice plants must also occur. Answer: Thank you so much for providing a hypothesis related to rice plant absorption. Actually, our hypothesis was impressing the loss of nutrients via the SW pathway rather than SbW percolation. In the rice ecosystems, the loss could be attributed to runoff, evaporation (nitrogen gases), and rice plant absorption. We seriously adopted your mentioned hypothesis to amend the explanation. The modification is as follows “This means that nutrient losses could occur through the SW runoff, evaporation (nitrogen gases) and rice plant absorption rather than leaching to SbW” 4.2 Comment 2: “N concentration loss” à ?? Answer: We removed “concentration” has been eliminated. It is as follows “N loss” 4.3 Comment 3: “ It is well-known that N-NH4+ in the rhizosphere area generally lowers the N-NO3- as rice prefers N-NH4+, up taking N-NH4+ faster than N-NO3- “ à not clear Answer: We rephrased the sentence in cohesion with the first sentence which is as follows: “ Our study found that N-NO3- concentrations showed no significant difference between the SW and SbW, while N-NH4+ in the SbW was consistently lower than in the SW (Ngan et al., 2021). This could be partly explained by rice plants preferring uptake of N-NH4+ rather than N-NO3-, resulting in higher N-NO3- concentration disclosed in the SbW”. 4.4. Comment 4: “a higher concentration of N-NO 3 − found below the soil surface (mostly in the rhizosphere) resulted in it being quickly transported to the soil surface” à correct? to the subsoil? Answer: Thank you for correcting the expression. It has been adopted. Revision is as follows “a higher concentration of N-NO 3 − found below the soil surface (mostly in the rhizosphere) resulted in it being quickly transported to the subsoil”. 4.5 Comment 5: This implied that the regression of organic matter mineralization/decomposition could occur slowly à not clear Answer: We agree with the reviewer that the expression did not clear. Thus, we simplified this sentence as follows: This implied that the regression of organic matter mineralization/decomposition could occur slowly “SOM plays an inevitable role in promoting nutrient availability and improving soil fertility. Our study found that SOM change was minor during rice growth. This could be partly explained by no organic matter sources being incorporated into the soil.” We look forward to hearing from you. Best regards, Authors."
}
]
}
] | 1
|
https://f1000research.com/articles/10-1145
|
https://f1000research.com/articles/11-1059/v1
|
16 Sep 22
|
{
"type": "Research Article",
"title": "Detection of SARS-CoV-2 in conjunctival secretion and tears in patients with COVID-19 in a tertiary care centre, South India. .",
"authors": [
"Rajesh R. Nayak",
"Sevitha Bhat",
"Ajay R Kamath",
"Anshul Chandak",
"Kanishk Khare",
"Rajesh R. Nayak",
"Ajay R Kamath",
"Anshul Chandak",
"Kanishk Khare"
],
"abstract": "Aims and objectives: Purpose of this study is to detect the presence of SAR-CoV-2 viral RNA in conjunctival secretions of COVID-19 patients and to compare the RT-PCR positivity rate for SARS-CoV-2 in conjunctival and nasopharyngeal swab. Materials and method: Eighty hospitalised COVID-19 patients whose nasopharyngeal swab tested positive for SARS-CoV-2 by RT-PCR were included in the study. Conjunctival swab was collected from eyes of these patients and sent for detection of SARS-CoV-2 by RT-PCR method. Results: Among the eighty patients, 51 (63.7%) were males and 29 (36.3%) were females. The mean age of the patients was 55.93 ± 16.59. Six patients had ocular manifestations. Eleven (13.75%) patients tested positive on conjunctival swab for SARS-CoV-2 viral RNA and only one of them had ocular manifestations out of the eleven. Conclusion: In our study the presence of SARS-CoV-2 in conjunctival secretions of COVID-19 patients was detected and this was not dependent on presence of ocular manifestations or duration of disease. Though the conjunctival positivity is lower compared to the nasopharyngeal swab sampling, ocular surface and secretions can be a potential route of viral transmission.",
"keywords": [
"Conjunctival swab",
"nasopharyngeal swab",
"SARS-CoV-2",
"COVID-19",
"RT-PCR"
],
"content": "Introduction\n\nAn outbreak of pneumonia of unknown cause was first reported in late December 2019 from Wuhan, China. A new variant of coronavirus temporarily called 2019 novel coronavirus (2019-nCoV) was found to be the culprit behind it.1 This non-segmented enveloped RNA virus of the Coronaviridae family was named ‘severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)’ as it resembled the virus causing the 2003 SARS outbreak, genetically, and the disease was termed as COVID-19 (Coronavirus disease).2,3\n\nClinically majority of patients have symptoms of fever, dry cough, and breathlessness while the less common symptoms include headache, fatigue, confusion, and diarrhoea. Though the majority of patients are asymptomatic or have mild to moderate influenza-like illness, severe cases of COVID-19 may present with a severe lung infection, acute respiratory or renal injury, septic shock, etc.4,5 Ground-glass opacity was a typical finding on the chest computed tomography which was seen in the majority of the patients. Decreased lymphocytes were another commonly seen finding on blood investigation.6 Alteration in taste and smell sensations has also been reported by patients with milder symptoms.7 Eye symptoms are also reported in a few patients which included watering, redness, itching, and discharge.8\n\nSARS-CoV-2 led to a global health crisis causing COVID-19. Worldwide a total of 220 million individuals have been diagnosed with the presence of the virus, and approximately 4.5 million people among them have been lost to COVID-19. In India alone, approximately 33 million individuals were found positive for SARS-CoV-2, and approximately half a million individuals have died. Though, the vaccination drive is at its height still the cases of COVID-19 are on the rise. The transmission of the coronavirus appears mainly through respiratory droplets and direct contact as the major routes for infection. Transmission through the gastrointestinal route, aerosol and eye secretions, is still to be further investigated. The possibility of transmission of the virus through ocular surfaces like the conjunctiva and cornea is still unproven.9–12 This highly contagious disease has presented as a medical challenge in terms of diagnosing and management because of the wide spectrum of disease symptoms with which a patient presents. RT-PCR (Reverse Transcription Polymerase Chain Reaction) test to look for SARS-CoV-2 in swabs taken from the nasopharyngeal area is still the gold standard for diagnosing COVID-19 disease.13\n\nThe ocular surface may be an inoculation site by direct touch or aerosols. From the conjunctiva, the virus may further spread to the cornea or may spread through the tears and secretions draining into the nasolacrimal duct to finally reach the nasopharynx. There have been few studies conducted worldwide demonstrating the presence of viruses in ocular secretions.\n\nIn our study, we aimed towards detecting SARS-CoV-2 in the conjunctival secretions and tears of COVID-19 affected individuals via reverse-transcriptase polymerase chain reaction (RT-PCR). The viral presence in the tears and ocular secretions may suggest another potential route of virus transmission. Furthermore, it will support the importance of avoiding touching of eyes and the use of protective eyewear among the doctors and even the general population at large.\n\n\nMethods\n\nStudy design and setting: It was a prospective cross-sectional study conducted in a tertiary care set up with COVID-19 care in the southern state of India between June 2021 and July 2021.\n\nThe study commenced with Institutional level ethical clearance from the Institutional Ethics Committee, Kasturba Medical College, Mangalore with the approval number IEC KMC MLR06-2021/191.\n\nPatients admitted with a positive nasopharyngeal swab for SARS-CoV-2 within a duration of 1 week, and age of more than 18 years. Patients with Covid-19 like symptoms but whose RT-PCR was negative for Covid-19 were excluded. Critically ill patients who couldn’t give consent were also excluded.\n\nPatients presenting complaints were noted along with any ocular complaints. The patient’s co-morbidities were recorded and clinical parameters on presentation like body temperature and oxygen saturation (SpO2) were also taken from the patient’s medical record. Day of nasopharyngeal swab collection was recorded and days between the nasopharyngeal swab collection and conjunctival swab collection was calculated for every patient. Written informed consent was obtained from patients before sample collection.\n\nA bedside ophthalmologist examination was done and the patient’s eyelids, adnexa, conjunctiva, and cornea were examined before collection of samples. The sample was collected under aseptic conditions by the same ophthalmologist who was donned in personal protective equipment. Samples were collected by using nylon flocked swabs by sweeping movement over the inferior fornix from the medial to the lateral direction. Sample from both eyes was collected for each patient using separate swab sticks without the use of any topical anaesthetic drops. Both swab sticks were then dipped into a single viral transport medium and transferred to the institutional microbiology lab, maintaining a proper cold chain at all times. The RTPCR was performed on the samples using the kit protocol and the results were recorded.\n\nAll the data was entered into an excel sheet and analyzed using IBM SPSS version 25. The continuous and categorical variables have been represented as mean ± standard deviation and frequency percentages respectively. The association between conjunctival swab positivity and variables like the severity of COVID, presence of symptoms such as fever, breathlessness, cough, fatigue, myalgia, diarrhoea, and ocular symptoms were analyzed using the chi-square test. Correlation between the duration of days after which conjunctival swab is tested positive from the day of nasopharyngeal swab positivity, and oxygen saturation was done using Pearson’s correlation and p<0.05 was considered significant.\n\n\nResults\n\nA total of 80 patients who were tested positive for SARS-CoV-2 on nasopharyngeal swabs were included in our study. Among them 51(63.7%) were males and 29(36.3%) were females. The mean age of the patients was 55.93 years with a standard deviation of 16.59 years. The average time interval between conjunctival swab collection and nasopharyngeal swab collections was similar in overall patients when compared to patients with conjunctival swab positivity with an average interval of 3.96±2.25 days and 3.45±2.73 days respectively. The mean of baseline parameters (like body temperature, respiratory rate, and oxygen saturation) of patients with conjunctival swab positivity was also similar to that of overall study patients (Table 1).\n\nThe presence of COVID-19 related symptoms did not show any variation in distribution among conjunctival swab-positive patients as compared to the whole study sample. Fever was the most common symptom among these patients seen in 70% of patients followed by cough (60%), breathlessness (52.5%), fatigue (36.3%), myalgia (18.8%), diarrhoea (8.8%), and ocular symptoms (7.5%) (Table 2). The association between Conjunctival swab positivity and the severity of COVID-19 infection is shown in Table 3.\n\nA total of 11 patients showed conjunctival swab positivity for SARS-CoV-2 by RT-PCR test. Among them, only one patient had ocular symptoms as compared to five patients complaining of ocular symptoms in whom conjunctival swab showed a negative result for the presence of the concerned virus. There was no correlation found between conjunctival swab positivity and the presence of ocular symptoms by using the chi-square test (Table 4).\n\nConjunctival swab positivity was not correlated to the time interval between conjunctival swab collection from that of nasopharyngeal swab collection as shown by Spearman's correlation. This shows conjunctival swab positivity is not affected by disease duration among COVID-19 patients (Table 5).\n\n\nDiscussion\n\nOne of the earliest studies conducted in China by Zang et al demonstrated SARS-CoV-2 on the ocular surface of 102 patients, among which only two patients tested positive for the RNA virus. Ping Wu et al also conducted a similar study and found two (5.2%) out of 28 patients who were positive for the virus on nasopharyngeal swabs. Similar studies were also performed worldwide to investigate ocular surface and secretions as the probable transmission source of SARS-CoV-2. K Kumar et al were among the first to study the conjunctival presence of SARS-CoV2 in the Indian population and found one (2.23%) conjunctival swab positive out of 45 samples studied.3\n\nIn our study, out of 80 nasopharyngeal swab-positive patients, 11(13.75%) patients have detected the concerned virus in their conjunctival secretions also. Our study showed a higher conjunctival swab positivity rate as compared to a few of the previously conducted studies.\n\nSimilar results were found by H Kaya et al, where they studied the prevalence of the virus among 32 COVID-19 patients with a positivity rate of 16% (five out of 32) was found. None of them showed conjunctivitis. Interestingly, at the time of conjunctival swab collection, a repeat nasopharyngeal swab was also sent. Out of five conjunctival swab-positive patients, two were tested negative on the nasopharyngeal swab test.14 This may indicate the possibility of viral transmission through ocular surface and secretions, even after nasopharyngeal swab results are negative.\n\nIn another study conducted among the Indian population from the Northern parts, found a higher prevalence of conjunctival positivity of 24% (18 patients) in a study population of 75 patients with moderate to severe COVID-19 disease without any ocular symptoms.11 Claudio Azzolini et al, also concluded a higher prevalence in a cohort of 91 patients, with 52 patients (57.1%) having conjunctival swab positivity for SARS-CoV-2.15\n\nThe patients included in our study had a mean age of 55.93 years which was comparable to the conjunctival swab positive group of patients, 56.36 years. The systemic symptoms in the majority of patients were fever (70%), cough (60%), and breathlessness (52.5%). The mean SpO2 of the patients in the conjunctival swab positive group was slightly lower (94%) as compared to the patients who were negative for the same (95%).\n\nVarious studies have been conducted to compare the ocular manifestations, mainly conjunctivitis, and compare it with the positivity rate of detection of the virus in their conjunctival secretions. Noemi Güemes-Villahoz et al, in Spain, studied 36 COVID-19 patients, with 18 patients each in conjunctivitis and non-conjunctivitis group, and found a similar prevalence of 5.5% (one patient) of conjunctival positivity in each group.16\n\nMahmoud H et al also studied 28 covid patients and found a higher conjunctival positivity prevalence rate of 28.57% (eight out of 28). Among ten patients with ocular symptoms, three tested positive on conjunctival swab while in the remaining 18 patients without ocular manifestations, five tested positive on the conjunctival swab for SARS-CoV-2.17 Therefore, the authors suggested the presence of the COVID-19 virus on the ocular surface or secretions is not affected by the presence of conjunctivitis.\n\nIn our study, a total of six patients had ocular manifestations. The main symptoms were redness and watery discharge. SARS-CoV-2 was detected in the conjunctival secretions in one patient with conjunctivitis. Other five patients who had ocular symptoms tested negative for the RNA virus on conjunctival swab sampling. Ten patients among the conjunctival positive group did not have any ocular manifestations.\n\nPrempal Kaur et al in a study conducted in northern India demonstrated RNA virus in conjunctival secretions of COVID 19 patients with or without ocular manifestations. The study included 60 COVID-19 patients each in two groups- one with ocular symptoms and the other without ocular symptoms (control). A similar positivity rate was found in both cases (18.33%) and control (16.66%) groups.18\n\nIn our study, we also evaluated the effect of duration between the nasopharyngeal swab to the conjunctival swab collection on the viral persistence in conjunctival secretions. The mean duration in our total sample of 80 patients was 3.96 days with a standard deviation of 2.25 days, while in the conjunctival swab positive patients it was 3.45 days (2.73±), with minimum and maximum duration being one and seven days respectively. This shows that detection of virus in conjunctival secretions may vary, and is not dependent on the duration. Thus, the chance of transmission of the RNA virus through the ocular surface is possible even after seven days of presentation, when most of the systemic symptoms of COVID-19 subside.\n\nTo study the ocular route of transmission of the virus, Wei Deng et al conducted an animal study on rhesus monkeys and found SARS-CoV-2 inoculation on the ocular surface can cause mild Covid-19 in these monkeys suggesting transmission of the virus through the ocular route is a possibility.19\n\nThe importance of protective eyewear can hence be ascertained and should be strictly practiced by doctors especially the ophthalmologist.\n\nOur study had the limitation of a small sample size. A multi-centric and a larger cohort may be needed to further validate the ocular route of transmission of the virus. Another limitation was that the conjunctival swab was collected only once. Our study showed that the virus was detected from the conjunctival secretions as early as day one to as late as day seven from nasopharyngeal swab positivity. Therefore, results may vary with multiple sampling done over a period of time.\n\nIn conclusion, our study demonstrated SARS-CoV-2 in conjunctival secretions of COVID-19 patients and is not dependent on the presence of ocular manifestations or duration of disease. Though the conjunctival positivity is lower compared to the nasopharyngeal swab sampling, ocular surface and secretions can be a potential route of viral transmission.\n\n\nAuthor contributions\n\n\n\n1. Dr Rajesh Nayak: Conceptualization, Data Curation, Formal Analysis, Funding Acquisition, Investigation,Methodology, Project Administration, Resources, Software Supervision, Validation, Visualization, Writing – Review & Editing\n\n2. Dr Sevitha Bhat - Data Curation, Formal Analysis, Investigation, Methodology, Project Administration, Supervision, Validation, Visualization, Writing – Review & Editing\n\n3. Dr Ajay R Kamath: Conceptualization, Funding Acquisition, Project Administration, Resources, Supervision, Validation, Visualization, Writing – Review & Editing\n\n4. Dr Anshul Chandak: Data Curation, Formal Analysis, Investigation, Methodology, Software, Validation, Visualization, Writing – Original Draft Preparation\n\n5. Dr Kanishk Khare: Data Curation, Formal Analysis, Investigation, Methodology, Software, Validation, Visualization, Writing – Original Draft Preparation",
"appendix": "Data availability\n\nDryad. Detection of SARS-CoV-2 in conjunctival secretion and tears in patients with COVID-19 in a tertiary care centre, South India. DOI: https://doi.org/10.5061/dryad.rn8pk0pdp 20\n\n\nAcknowledgments\n\nThe authors are grateful to Manipal Academy of Higher education for the support.\n\n\nReferences\n\nNovel Coronavirus (2019-nCoV) Situation Report JANUARY 2020, World Health Organisation.Reference Source\n\nLi G, Fan Y, Lai Y, et al.: Coronavirus infections and immune responses. Vol. 92, Journal of Medical Virology. John Wiley and Sons Inc.;2020; 424–432.\n\nNaming the coronavirus disease (COVID-19) and the virus that causes it.World Health Organisation.Reference Source\n\nHuang C, Wang Y, Li X, et al.: Clinical features of patients infected with 2019 novel coronavirus in Wuhan, China. Lancet. 2020 Feb 15; 395(10223): 497–506. PubMed Abstract | Publisher Full Text\n\nChen N, Zhou M, Dong X, et al.: Epidemiological and clinical characteristics of 99 cases of 2019 novel coronavirus pneumonia in Wuhan, China: a descriptive study. Lancet. 2020 Feb 15; 395(10223): 507–513. PubMed Abstract | Publisher Full Text\n\nGuan W, Ni Z, Hu Y, et al.: Clinical Characteristics of Coronavirus Disease 2019 in China. N. Engl. J. Med. 2020 Apr 30; 382(18): 1708–1720. PubMed Abstract | Publisher Full Text\n\nSpinato G, Fabbris C, Polesel J, et al.: Alterations in Smell or Taste in Mildly Symptomatic Outpatients with SARS-CoV-2 Infection. Vol. 323, JAMA - Journal of the American Medical Association. American Medical Association;2020; 2089–2091.\n\nWu P, Duan F, Luo C, et al.: Characteristics of Ocular Findings of Patients With Coronavirus Disease 2019 (COVID-19) in Hubei Province, China. JAMA Ophthalmol. 2020 May 1; 138(5): 575–578. Publisher Full Text\n\nGe H, Wang X, Yuan X, et al.: The epidemiology and clinical information about COVID-19. Vol. 39, European Journal of Clinical Microbiology and Infectious Diseases. Springer;2020; 1011–1019.\n\nChen Y, Guo Y, Pan Y, et al.: Structure analysis of the receptor binding of 2019-nCoV. Biochem. Biophys. Res. Commun. 2020 Apr 23; 525(1): 135–140. PubMed Abstract | Publisher Full Text\n\nMatos AG, Sarquis IC, Santos AAN, et al.: COVID-19: risk of ocular transmission in health care professionals. Rev. Bras. Med. Do Trab. 2021; 19(01): 82–87. PubMed Abstract | Publisher Full Text\n\nSun J, Zhu A, Li H, et al.: Isolation of infectious SARS-CoV-2 from urine of a COVID-19 patient.2020.\n\nGoudouris ES: Laboratory diagnosis of COVID-19. Vol. 97, Jornal de Pediatria. Elsevier Editora Ltda;2021; 7–12.\n\nKaya H, Çalışkan A, Okul M, et al.: Detection of SARS-CoV-2 in the tears and conjunctival secretions of Coronavirus disease 2019 patients. J. Infect. Dev. Ctries. 2020 Sep 1; 14(9): 977–981. PubMed Abstract | Publisher Full Text\n\nAzzolini C, Donati S, Premi E, et al.: SARS-CoV-2 on Ocular Surfaces in a Cohort of Patients with COVID-19 from the Lombardy Region, Italy. JAMA Ophthalmol. 2021; 139: 956–958. Publisher Full Text\n\nGüemes-Villahoz N, Burgos-Blasco B, Arribi-Vilela A, et al.: Detecting SARS-CoV-2 RNA in conjunctival secretions: Is it a valuable diagnostic method of COVID-19?. J. Med. Virol. 2021 Jan 1; 93(1): 383–388. PubMed Abstract | Publisher Full Text\n\nMahmoud H, Ammar H, El RA, et al.: Assessment of coronavirus in the conjunctival tears and secretions in patients with SARS-cov-2 infection in sohag province, Egypt. Clin Ophthalmol. 2020; 14: 2701–2708. PubMed Abstract | Publisher Full Text\n\nKaur P, Sehgal G, Shailpreet, et al.: Evaluation and comparison of conjunctival swab polymerase chain reaction results in SARS-CoV-2 patients with and without ocular manifestations. Indian J. Ophthalmol. 2021 Aug 1; 69(8): 2211–2214. Publisher Full Text\n\nDeng W, Bao L, Gao H, et al.: Ocular conjunctival inoculation of SARS-CoV-2 can cause mild COVID-19 in rhesus macaques. Nat. Commun. 2020 Sep 2; 11(1): 1–7. Publisher Full Text\n\nNayak R, et al.:Detection of SARS-CoV-2 in conjunctival secretion and tears in patients with COVID-19 in a tertiary care centre, South India, Dryad. Dataset. 2022. Publisher Full Text"
}
|
[
{
"id": "150606",
"date": "28 Sep 2022",
"name": "Anugraha Mathew",
"expertise": [
"Reviewer Expertise Microbiology",
"Molecular Biology"
],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe manuscript by Nayak et al. is original, reads well, and is of significant interest to clinicians, patients and policymakers. The authors describe the prevalence of coronavirus in the conjunctival secretions, regardless of specific ocular manifestations. Although SARS-CoV-2 conjunctivitis is rare in relation to the magnitude and prevalence of other manifestations and complications caused by the virus, the ophthalmic route of disease transmission is possible and hence this study is relevant and adds to the current knowledge of COVID-19 testing and prevention measures.\nMajor comments\nSince the objective of the study includes a comparison of the RT-PCR positivity rate for SARS-CoV-2 in conjunctival and nasopharyngeal swabs, it would be ideal to include the Ct values obtained with RT-PCR from both the samples. These values would also be a good indicator of the viral load in both samples and provide additional information to the current literature on the topic.\n\nThe authors conclude that ocular surface and secretions can be a potential route of viral transmission. Since ACE2 receptors serve as the entry point of the viral particle, a comment or discussion on the presence of these receptors on the ocular surface would be an additional point to support this hypothesis.\n\nMinor comments Introduction\nLine 1, 2nd paragraph: \"Clinically majority of patients have symptoms of fever, dry cough, and breathlessness while the less common symptoms include headache, fatigue, confusion, and diarrhoea.\" Maybe modified as \"Most patients have symptoms such as fever, dry cough, and breathlessness while the less common symptoms include headache, fatigue, confusion, and diarrhoea.\"\n\nLine 4, 3rd paragraph: \"The transmission of the coronavirus appears mainly through respiratory droplets and direct contact as the major routes for infection.\" Maybe modified as \"According to current evidence, COVID-19 virus is primarily transmitted between people through respiratory droplets and contact routes.\"\n\nLine 5, 3rd paragraph: \"Transmission through the gastrointestinal route, aerosol and eye secretions, is still to be further investigated. The possibility of transmission of the virus through ocular surfaces like the conjunctiva and cornea is still unproven.\" Maybe modified as \"The possibility of transmission through the gastrointestinal route, aerosol and eye secretions still needs further investigation. Likewise, the transmission of the virus through ocular surfaces like the conjunctiva and cornea is still unproven.\"\n\nLine 7, 3rd paragraph: \"This highly contagious disease has presented as a medical challenge...\" Maybe modified as \"This highly contagious disease represents a medical challenge...\"\n\nIs the work 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": "8832",
"date": "29 Sep 2022",
"name": "SEVITHA BHAT",
"role": "Author Response",
"response": "Reply to Major comments 11 of the patients with conjunctival swab positivity had the Ct values for the E and RdRp genes in the range of 25-30. The Ct values of the same patients in the nasopharyngeal swabs were in the range of 15-20. Though Ct value is not an estimate of the viral load, the values were more in the conjunctival swabs compared to the nasopharyngeal swabs. Previous studies have reported the presence of ACE2 in the conjunctiva, limbus, and cornea, reemphasizing the fact that conjunctiva is susceptible to SARS-CoV-2 infection.20 Reference 20 - Zhou L, Xu Z, Castiglione GM, Soiberman US, Eberhart CG, Duh EJ. ACE2 and TMPRSS2 are expressed on the human ocular surface, suggesting susceptibility to SARS-CoV-2 infection. Ocul Surf. 2020 Oct;18(4):537-544. Reply to Minor comments Line 1, 2nd paragraph: please modify the existing sentence as \"Most patients have symptoms such as fever, dry cough, and breathlessness while the less common symptoms include headache, fatigue, confusion, and diarrhoea.\" Line 4, 3rd paragraph: please modify the existing sentence as \"According to current evidence, COVID-19 virus is primarily transmitted between people through respiratory droplets and contact routes.\" Line 5, 3rd paragraph: please modify the existing sentence as \"The possibility of transmission through the gastrointestinal route, aerosol and eye secretions still needs further investigation. Likewise, the transmission of the virus through ocular surfaces like the conjunctiva and cornea is still unproven.\" Line 7, 3rd paragraph: please modify the existing sentence as \"This highly contagious disease represents a medical challenge\""
}
]
}
] | 1
|
https://f1000research.com/articles/11-1059
|
https://f1000research.com/articles/12-15/v1
|
05 Jan 23
|
{
"type": "Research Article",
"title": "Trends in pancreatic cancer incidence and mortality in the United States from 2000 to 2019; a SEER based study",
"authors": [
"Oluseyi Abidoye",
"Young Min Cho",
"Sheena Bhushan",
"Comfort Adewunmi",
"Hasan Choudhury",
"Young Min Cho",
"Sheena Bhushan",
"Comfort Adewunmi",
"Hasan Choudhury"
],
"abstract": "Background: The annual incidence and mortality rates of pancreatic cancer has been rising. We analyzed pancreatic cancer trends by demographics and geographic location. Methods: Data was obtained from the Surveillance, Epidemiology, and End Results (SEER) registry 17 were available to assess trends of pancreatic cancer incidence and mortality from 2000 to 2019. Results: The pancreatic cancer incidence and mortality rates consistently increased over time from 2000 to 2019. Incidence of pancreatic cancer was highest in males (14; 95% Cl, 13.9,14.1) and blacks (15.4 95% Cl,15.2, 15.6). The incidence was highest in Alaska (16.2; 95% Cl, 14.1, 18.5), and lowest in Utah (10.7; 95% Cl, 10.4, 11.0). Incidence increased at 0.85% per year (95% Cl, 0.76, 0.92; P < .001). Overall incidence increased significantly for all sex, race, stage sub-groups except for Blacks (APC of 0.04%; 95% Cl, -0.14, 0.22; P = 0.649), and American Indian/Alaska natives (APC of -0.22 %; 95% Cl, -1.33, 0.89; P= 0.679). There was an overall significant increase in incidence across all states except for Alaska. Incidence-based mortality of pancreatic cancer was highest in males (12.6; 95% Cl, 12.5,12.7) and blacks (14.1; 95% Cl, 13.9, 14.3). Mortality was highest in Alaska (15.1; 95% Cl, 13.1,17.3), and lowest in Utah (9.6; 95% Cl, 9.3,9.9). Mortality increased at 0.91% per year (95% Cl, 0.32, 1.50; P = .005). The overall incidence-based mortality increased significantly only in the males APC, 0.95% (95% Cl, 0.36, 1.54; P = 0.003), females APC 1.18 (95% Cl, 0.47, 1.89; P = 0.002) and whites APC 1.05% (95% Cl, 0.45, 1.66; P = 0.002). Furthermore, there was an overall significant increase in incidence-based mortality across all states except for Alaska, Hawaii and Washington. Conclusion: Pancreatic cancer incidence and mortality rates increased overall but differed by demographics and geographic location.",
"keywords": [
"Pancreatic Cancer",
"Pancreatic cancer incidence",
"Pancreatic Incidence-based mortality",
"SEER database",
"Pancreatic cancer risk factors"
],
"content": "Introduction\n\nPancreatic cancer (PC) is one of the deadliest malignancies accounting for the third leading cause of cancer deaths in the United States in both male and females1–3 and seventh leading cause of cancer deaths worldwide.3 The American Cancer Society’s estimates about 62,210 people (32,970 men and 29,240 women) will be diagnosed with pancreatic cancer and about 49,830 people (25,970 men and 23,860 women) will die of pancreatic cancer in the United States by 2022.1 Pancreatic cancer accounts for about 3% of all cancers in the US and about 7% of all cancer deaths. It is one of the most fatal malignancies evidenced by a five-year survival rate of 11%.4 In terms of sex and race, pancreatic cancer rates have been reported to be higher in among male and black population.\n\nPancreatic cancer can be sub-grouped based on the histology. About 95% of malignant neoplasms of the pancreas originate from the exocrine elements with ductal adenocarcinoma accounting for about 85% of all pancreatic neoplasms.5 Neuroendocrine tumors such as insulinoma, gastrinoma, glucagonoma, somatostatinoma, and non-functional islet cells tumors are less common histological types of pancreatic cancer.5 Approximately 60 to 70 percent of exocrine pancreatic cancers are localized to the head of the pancreas, while 20 to 25 percent are in the body/tail and the remainder involve the whole organ.6\n\nStudies have identified modifiable risk factors have been associated with Pancreatic cancer such as Overweight, Obesity, Tobacco use, Cigarette smoking and diabetes especially pancreatic adenocarcinoma7,8,9 with limited data on risk factors associated with other pancreatic cancers.10\n\nTypically, patients are initially symptom-free until tumor progression when they present with non-specific symptoms such as fatigue, abdominal pain, jaundice, and weight loss.11 Diagnosis most cases are incidental in nature with nonspecific imaging studies. Most times, diagnosis is missed because patients have an early-stage disease.12 Treatment entails surgery, chemotherapy and radiotherapy depending on the staging and extent of disease, with most cases treatment focused on symptom relief and improving survival as there are no definitive treatments for advanced disease.11,12\n\nThe lack of screening tests and high fatality makes pancreatic cancer a tremendous public health burden for the United States.12 Based on the high fatality and absence screening tests, a need for further research focusing on interventions directed at disease prevention, treatment, and patient outcomes.\n\nA trend analysis of incidence and mortality rate of pancreatic cancer is focused on highlighting the burden of pancreatic cancer and emphasize the importance of formulating preventive strategies geared at reducing pancreatic cancer incidence and mortality. Our study focused on obtained data from National Cancer Institute (NCI) from the Surveillance, Epidemiology, and End Results (SEER) database13 and highlights the incidence and incidence-based mortality trends from 2000-2019.\n\nThe SEER program of the NCI has been involved in collecting data on cancer epidemiology.13 Several studies have analyzed and assessed trends in pancreatic cancer rates.14,15 However, most reports have varied in conclusions with recent data. Most of these reports used SEER database as their only database. Our study further focused on stratifying pancreatic cancer incidence rates and incidence mortality-based rates based on age, sex, race and state.\n\n\nMethods\n\nWe used SEER*stat software 8.4.0.116 to obtain data of pancreatic cancer cases diagnosed from 2000 to 2019 from SEER eighteen registries. “Incidence - SEER Research Plus Data, 17 Registries, Nov 2021 Sub (2000-2019). The SEER 17 data is based on 26.5% of the U.S. population based on 2010 census. The study included patients diagnosed with pancreatic cancer from 2000 to 2019; We used International Classification of Disease (ICD)-0-2 and ICD-0-3 for coding for pancreatic cancer.17,18 We excluded cases whose diagnosis relied only on autopsy or death certificates. We focused on the following variables: sex, race, age and stage at diagnosis, site of the tumor within the pancreas and geographical location.\n\nThe rates are available by expanded race/ethnicity of cases diagnosed, including white, black, Asian/Pacific Islander, and American Indian/Alaskan Native and Hispanic ethnicity. SEER 17 also includes adjustments for areas impacted by hurricanes Katrina and Rita. It contains a record for each of 8,131,919 tumors. The registries included in SEER 17 are Alaska Natives, San Francisco Oakland SMSA, Connecticut, Hawaii, Iowa, New Mexico, Seattle (Puget Sound), Utah, Atlanta, (Metropolitan, San Jose-Monterey, Los Angeles, Rural Georgia, California excluding San Francisco, San Jose Monterey, Los Angeles, Kentucky, Louisiana, New Jersey, Greater Georgia.\n\nWe calculated two main outcomes: incidence and incidence-based mortality rates. All rates were adjusted to the 2000 US Standard population and expressed by 100, 000 person-years. These rates were calculated from 2000 to 2019 according to demographic and tumor characteristics. Incidence-based mortality rates were calculated as the number of pancreatic cancer deaths among cases diagnosed over person-time at risk among people in the SEER areas. We then calculated the Annual percentage changes (APC) of incidence and incidence-based mortality rates over the study period according to the baseline demographic and tumor characteristics.\n\nWe used SEER*stat software 8.4.0.1 to calculate the incidence and incidence-based mortality rates. We also used the NCI’s Joinpoint Regression Program, version 4.9.1.019,20 to calculate the APCs. The Joinpoint Regression software uses t-tests to determine if APCs were statistically significant from zero; a difference was noted to be statistically significant when P < 0.05. The software analyzed rates over time and detected significant changes in the APCs, then selected the best model with the minimum number of joinpoints. All statistical tests were two-sided.\n\n\nResults\n\nWe obtained data of 206,968 cases with pancreatic cancer during 2000 – 2019 period. (Table 1). Majority of these patients were white (148,321 subjects [80.87%]), Male (92,438 ejects 50.40%]), older than 60 (152,764 subjects [83.29%]) and had metastatic disease at diagnosis (86,578 subjects [41.83%]). In terms of geographic location, California state had the highest subjects of 75,532 (41.18%).\n\na Cases reported from the SEER registry\n\nb Rates were calculated as the number of cases per 100,000 person-years and age-adjusted to the 2000 US standard population\n\nc Rates for patients with unknown race could not be calculated, as ‘race’ is a population variable and must be known to calculate rates\n\nd Rates were calculated using Using SEER registry variable between 2000 – 2018 for all states, with Georgia and California subdivided based on region\n\ne Using SEER Summary stage 2000\n\nWe also reviewed data of 183,406 cases of patients who died from pancreatic cancer based on incidence-based mortality analysis from 2000 – 2019 period (Table 2). Most of the patients were male (92,438 subjects [50.40%]), White (148,321 subjects [80.87%]), older than 60 (152,764 subjects [83.29%]) and had a metastatic disease (87,142 subjects [40.91%]). Figure 1 shows the incidence and incidence-based mortality trends for Pancreatic Cancer from 2000-2019.\n\na Cases reported from the SEER registry\n\nb Rates were calculated as the number of cases per 100,000 person-years and age-adjusted to the 2000 US standard population\n\nc Rates for patients with unknown race could not be calculated, as ‘race’ is a population variable and must be known to calculate rates\n\nd Rates were calculated using Using SEER registry variable between 2000 – 2018 for all states, with Georgia and California subdivided based on region\n\ne Using SEER Summary stage 2000\n\n*Indicates that the Annual percentage Change (APC) is significantly different from zero at the alpha =0.05 level.\n\nThe overall pancreatic incidence during the study period was 12.3 per 100,000 person-years (95% confidence interval [Cl], 12.3, 12.4). Incidence of pancreatic cancer was highest in males (14; 95% Cl, 13.9,14.1), black subjects (15.4 95% Cl,15.2, 15.6), and people older than 60 years (60.2; 95% Cl, 59.9, 60.5). In terms of geographical location, the incidence was highest in Alaska (16.2; 95% Cl, 14.1, 18.5), and lowest in Utah (10.7; 95% Cl, 10.4, 11.0) compared to other states. Based on the stage at diagnosis, distant stage had the highest incidence (5.1; 95% Cl, 5.1, 5.2). (Table 1)\n\nOver the study period, pancreatic incidence rates increased at 0.85% per year (95% Cl, 0.76, 0.92; P < .001). Rates did increase but were not significant during periods 2000-2003; APC, 0.71% (95% Cl, -0.56, 2.0 P = 0.248) and 2003-2006; APC, 1.74% (95% Cl, -0.71, 4.26, P= 0.149, but significantly increased by 0.68% (95% Cl, 0.56, 0.80; P < 0.001) per year during 2006 – 2019. The overall incidence rates increased significantly for all sex, race, age at diagnosis and stage at diagnosis except for Black subjects (APC of 0.04%; 95% Cl, -0.14, 0.22; P = 0.649), and American Indian/Alaska native subjects (APC of -0.22 %; 95% Cl, -1.33, 0.89; P= 0.679). Furthermore, there was an overall significant increase in incidence rates across all states except for Alaska. (file 3) Table 3 describes the pancreatic cancer incidence trends from 2000 – 2019 by sex, race, stage and age at diagnosis. Table 5 pancreatic cancer incidence trends from 2000 – 2019 by geographical location. Figure 2 shows the incidence trends based on sex, race and stage; Figure 3 shows the incidence trends based on state.\n\n*Indicates that the Annual percentage Change (APC) is significantly different from zero at the alpha =0.05 level.\n\n*Indicates that the Annual percentage Change (APC) is significantly different from zero at the alpha =0.05 level.\n\nThe overall pancreatic incidence-based mortality during the study period was 11 per 100,000 person-years (95% Cl, 10.9,11). Incidence-based mortality of pancreatic cancer was highest in males (12.6; 95% Cl, 12.5,12.7), black subjects (14.1; 95% Cl, 13.9, 14.3), and people older than 60 years (56.1; 95% Cl, 55.8,56.4). In terms of geographical location, incidence-based mortality was highest in Alaska (15.1; 95% Cl, 13.1,17.3), and lowest in Utah (9.6; 95% Cl, 9.3,9.9) compared to other states. Based on the stage at diagnosis, distant staging (5.2; 95% Cl, 5.1, 5.2) had the highest incidence-based mortality compared to stage sub-groups. (Table 2).\n\nOver the study period, pancreatic incidence-based mortality rates increased at 0.91% per year (95% Cl, 0.32, 1.50; P = .005). There was a significant steep increase from 2000 – 2002 period; APC, 21.57% (95% Cl, 15.88, 27.88 P <0.001), followed by a gradual increase in 2002-2007; APC, 1.02 % (95% Cl, -0.12, 2.17, P= 0.075) which was not significant; However, 2007 – 2019 period increased by 0.27% per year which was significant (95% Cl, 0.07, 0.47; P = 0.013). The overall incidence-based mortality rates increased significantly only in the male subjects APC, 0.95% (95% Cl, 0.36, 1.54; P = 0.003), female subjects APC 1.18 (95% Cl, 0.47, 1.89; P = 0.002) and white subjects APC 1.05% (95% Cl, 0.45, 1.66; P = 0.002) and age at diagnosis. Even though there was an increase in the other subgroups, the increase was not significant. Furthermore, there was an overall significant increase in incidence-based mortality rates across all states except for Alaska, Hawaii and Washington. Table 4 describes the pancreatic cancer incidence-based mortality trends from 2000 – 2019 by sex, race, age at diagnosis and stage. Table 6 pancreatic cancer incidence-based mortality trends from 2000 – 2019 by geographical location.\n\nFigure 4 shows the incidence-based mortality trends based on sex, race and stage at diagnosis; Figure 5 shows the incidence-based mortality trends based on state.\n\n*Indicates that the Annual percentage Change (APC) is significantly different from zero at the alpha =0.05 level.\n\n*Indicates that the Annual percentage Change (APC) is significantly different from zero at the alpha =0.05 level.\n\n\nDiscussion\n\nOur study highlighted on the trends of incidence and mortality rates of pancreatic cancer in the United States using a single comprehensive registry system from 2000 to 2019. Incidence rates of pancreatic carcinomas consistently increased over time from 2000 to 2019 but differed by gender, race, staging and geographical location. Pancreatic cancer incidence rates increased in all sex, race, stage and age at diagnosis except for Blacks and American Indian/Alaskan Native individuals, although rates of pancreatic cancer consistently were higher overall among the Black population. Such increases in rates of pancreatic cancer have also been observed internationally.21,22 Based on location, the incidence rates increased in most states except for Alaska and Hawaii; Alaska had the overall highest rates of pancreatic cancer incidence per state. Several studies have also reported increases in rates based on location as well.\n\nThe Pancreatic incidence-based mortality rates increased consistently in only males, females and white population; while with the other subgroups their incidence-based mortality rates remained relatively the same. Incidence-based mortality of pancreatic cancer was highest in males, black population and in Alaska. Regarding location, the incidence-based mortality rates increased across all states except for Alaska, Hawaii and Washington.\n\nThere are have been reported risk factors such as Obesity, Overweight, Tobacco smoking, Diabetes and Chronic pancreatitis that have been associated with Pancreatic Cancer.7,8,9,10 Tobacco smoking is a one of the established risk factors of Pancreatic Cancer.23,24 Several studies have showed a causal relationship with tobacco smoking especially with the number of cigarettes smoked with a study showing risk of pancreatic cancer decreasing by about 40% in two years after smoking cessation.\n\nVanessa L Gordon-Dseagu et al15 reported a decrease in incidence of pancreatic cancer from 1974 through 1990’s partly due to the drop in the rate of cigarette smoking in the mid-1960s. There has been a notable decline in smoking rates among all age groups, gender and racial/ethnic groups in recent decades but regardless of this decline; pancreatic cancer incidence has been rising steadily. This increase incidence of pancreatic cancers may be related to an increasing prevalence of obesity and overweight. The rate of prevalence of obesity and overweight continues to increase in the United states.25Studies have attributed steady increase in pancreatic cancer incidence to prevalence of obesity and overweight.9\n\nBased on a recent study25, prevalence rates of overweight/obesity have been highest among Male and Black populations; these rates reflect higher pancreatic cancer incidence rates in among these groups. Furthermore, Asian populations have lower pancreatic cancer rates and this may be due to their the low prevalence of obesity and overweight compared with other racial groups.\n\nDiabetes Mellitus has been reported to be associated with pancreatic cancer.8,27–29 It has been reported that abnormalities in glucose metabolism, insulin resistance and deficiency have been associated with pancreatic cancer. A recent meta-analysis reported a higher relative risk for pancreatic cancer associated with diabetes compared to the non-diabetics.26 These studies are further supported with the increase in prevalence rates of diabetes. The prevalence of diabetes was higher in Male population compared to female population as well as Black population compared other racial groups.25 This could be a contributory factor associated with the increase trends of pancreatic cancer incidence.\n\nOverall, the incidence and mortality rates of pancreatic cancer increased consistently from 2000-2019. Our observations are consistent with similar studies done during different time periods. There was a study that focused on pancreatic adenocarcinoma from 1973 to 2014, reporting similar results with our study. Our study focused on all pancreatic cancers from 2000 to 2019. As noted from previous study, there was an overall increase in the incidence and incidence-based mortality rates of pancreatic cancers during the study group.\n\nOne of the strengths of our study was use of large population-based database, with SEER 21 covering approximately 26.5% of the US population. This allowed us the opportunity to analyze the recent pancreatic cancer rates. Our study had several limitations due to limited available date from SEERs. Because of the limitations with SEERs database, we were unable to compare factors associated with pancreatic cancer such as lifestyle habits or co-morbidities which prevented us from identifying a direct association between such factors and the incidence and mortality of pancreatic cancer.\n\n\nConclusion\n\nPancreatic cancer incidence and mortality rates have been increasing in recent decades in the USA. We analyzed incidence and mortality rates by sex, race, age, staging, and geographic location. The increasing incidence trends may be attributed to an increase in known risk factors such as smoking, obesity, and diabetes; Health professionals and health policymakers should make a conscious effort in reducing these factors by creating initiatives and efforts at curbing these factors such as awareness, advocating for healthy lifestyles. The increasing mortality trends can be correlated to multiple factors such as late detection and diagnosis. Improvements in early detection, diagnosis and management may reduce the mortality rate over time.\n\nAdditional file 1, shows the incidence rates in each individual year from 2000 to 2019.\n\nAdditional file 2 shows pancreatic cancer incidence-based mortality rates in each individual year from 2000 to 2019.\n\n\nContribution\n\nAbidoye O: Conceptualization, Investigation, Methodology, Project Administration, Resources, Software, Supervision, Validation, Visualization, Writing – Original Draft Preparation, Writing – Review & Editing; Cho Y: Supervision, Writing – Original Draft Preparation, Writing – Review & Editing; Bhushan S: Writing – Original Draft Preparation; Adewunmi C: Validation; Choudhury H: Validation\n\n\nConflict-of-interest disclosure\n\nThe authors declare no financial interests.",
"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\nReference\n\nCancer Facts & Figures: American Cancer Society (ACS), Atlanta, Georgia.2022; 2022.\n\nHowlader N, Noone AM, Krapcho M, et al.: SEER Cancer Statistics Review, 1975-2018. Bethesda, MD:National Cancer Institute.\n\nSung H, Ferlay J, Siegel RL, et al.: Global Cancer Statistics 2020: GLOBOCAN Estimates of Incidence and Mortality Worldwide for 36 Cancers in 185 Countries. CA Cancer J. Clin. 2021 May; 71(3): 209–249. Epub 2021 Feb 4. PubMed Abstract | Publisher Full Text\n\nSiegel RL, Miller KD, Fuchs HE, et al.: Cancer Statistics, 2021. CA Cancer J. Clin. 2021 Jan; 71(1): 7–33. Epub 2021 Jan 12. Erratum in: CA Cancer J. Clin. 2021 Jul;71(4):359. PubMed Abstract | Publisher Full Text\n\nZhou J, Enewold L, Stojadinovic A, et al.: Incidence rates of exocrine and endocrine pancreatic cancers in the United States. Cancer Causes Control. 2010 Jun; 21(6): 853–861. Epub 2010 Feb 25. Erratum in: Cancer Causes Control. 2011 Sep;22(9):1353. PubMed Abstract | Publisher Full Text\n\nModolell I, Guarner L, Malagelada JR: Vagaries of clinical presentation of pancreatic and biliary tract cancer. Ann. Oncol. 1999; 10 Suppl 4: 82–84. PubMed Abstract | Publisher Full Text\n\nSiegel RL, Jacobs EJ, Newton CC, et al.: Deaths due to cigarette smoking for 12 smoking-related cancers in the United States. JAMA Intern. Med. 2015; 175: 1574–1576. PubMed Abstract | Publisher Full Text\n\nKamisawa T, Wood LD, Itoi T, et al.: Pancreatic cancer. Lancet Lond Engl. 2016; 388: 73–85. Publisher Full Text\n\nStolzenberg-Solomon RZ, Schairer C, Moore S, et al.: Lifetime adiposity and risk of pancreatic cancer in the NIH-AARP Diet and Health Study cohort. Am. J. Clin. Nutr. 2013; 98: 1057–1065. PubMed Abstract | Publisher Full Text\n\nIlic M, Ilic I: Epidemiology of pancreatic cancer. World J. Gastroenterol. 2016; 22: 9694–9705. PubMed Abstract | Publisher Full Text\n\nMcGuigan A, Kelly P, Turkington RC, et al.: Pancreatic cancer: A review of clinical diagnosis, epidemiology, treatment and outcomes. World J. Gastroenterol. 2018 Nov 21; 24(43): 4846–4861. PubMed Abstract | Publisher Full Text | Free Full Text\n\nZhang L, Sanagapalli S, Stoita A: Challenges in diagnosis of pancreatic cancer. World J. Gastroenterol. 2018 May 21; 24(19): 2047–2060. PubMed Abstract | Publisher Full Text | Free Full Text\n\nSurveillance Research Program, NCIs Division of Cancer Control and Population Sciences: SEER Registries.20 march 2021. SEER*Stat Databases: November 2019 Submission (cancer.gov)\n\nAli H, Pamarthy R, Vallabhaneni M, et al.: Pancreatic cancer incidence trends in the United States from 2000-2017: Analysis of Surveillance, Epidemiology and End Results (SEER) database. F1000Res. 2021 Jul 2; 10: 529. PubMed Abstract | Publisher Full Text | Free Full Text\n\nGordon-Dseagu VL, Devesa SS, Goggins M, et al.: Pancreatic cancer incidence trends: evidence from the Surveillance, Epidemiology and End Results (SEER) population-based data. Int. J. Epidemiol. 2018 Apr 1; 47(2): 427–439. PubMed Abstract | Publisher Full Text | Free Full Text\n\nSurveillance Research Program, National Cancer Institute: SEER*Stat software. version 8.4.0.1.\n\nPercy C, Van Holten V, Muir C: ICD- O International Classification of Diseases for Oncology. 2nd ed.Geneva:World Health Organization.1990.Reference Source\n\nFritz A, Percy C, Jack A, et al.: ICD-O International Classification of Diseases for Oncology. 3rd ed.Geneva:World Health Organization.Reference Source\n\nNational Cancer Institute, Statistical Methodology and Applications Branch, Surveillance Research Program: Joinpoint Regression Program, Version 4.9.1.0—April 2022.\n\nKim HJ, Fay MP, Feuer EJ, et al.: Permutation tests for Joinpoint regression with applications to cancer rates. Stat. Med. 2000; 19: 335–351. PubMed Abstract | Publisher Full Text\n\nLin Q-J: Current status and progress of pancreatic cancer in China. World J. Gastroenterol. 2015; 21: 7988–8003. PubMed Abstract | Publisher Full Text\n\nWong MCS, Jiang JY, Liang M, et al.: Global temporal patterns of pancreatic cancer and association with socioeconomic development. Sci. Rep. 2017; 7: 3165. PubMed Abstract | Publisher Full Text\n\nStolzenberg-Solomon RZ, Amundadottir LT: Epidemiology and inherited predisposition for sporadic pancreatic adenocarcinoma. Hematol. Oncol. Clin. North Am. 2015; 29: 619–640. PubMed Abstract | Publisher Full Text\n\nSilverman DT, Hoover RN, Brown LM, et al.: Why do Black Americans have a higher risk of pancreatic cancer than White Americans? Epidemiol. Camb. Mass. 2003; 14: 45–54. PubMed Abstract | Publisher Full Text\n\nArispe IE, Gindi RM, Madans JH: Health, United States, 2019. National Center for Health Statistics (U.S.);2021.Reference Source\n\nBatabyal P, Vander Hoorn S, Christophi C, et al.: Association of diabetes mellitus and pancreatic adenocarcinoma: a meta-analysis of 88 studies. Ann. Surg. Oncol. 2014; 21(7): 2453–2462. PubMed Abstract | Publisher Full Text\n\nCarreras-Torres R, Johansson M, Gaborieau V, et al.: The Role of Obesity, Type 2 Diabetes, and Metabolic Factors in Pancreatic Cancer: A Mendelian Randomization Study. J. Natl. Cancer Inst. 2017; 109(9): djx012. Publisher Full Text\n\nMichaud DS, Liu S, Giovannucci E, et al.: Dietary sugar, glycemic load, and pancreatic cancer risk in a prospective study. J. Natl. Cancer Inst. 2002; 94(17): 1293–1300. Publisher Full Text\n\nWolpin BM, Bao Y, Qian ZR, et al.: Hyperglycemia, insulin resistance, impaired pancreatic β-cell function, and risk of pancreatic cancer. J. Natl. Cancer Inst. 2013; 105(14): 1027–1035. PubMed Abstract | Publisher Full Text"
}
|
[
{
"id": "212936",
"date": "24 Oct 2023",
"name": "Haleh Amirian",
"expertise": [
"Reviewer Expertise Clinical research with focus on pancreatic cancer"
],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nWell-written and informative retrospective study using SEER database looking at incidence and incidence-based mortality of pancreatic cancer through the years and among different racial groups, genders, stages, and states in the US. However, the results has not been fully discussed in this paper.\nWith regards to incidence, it has been increased throughout the years, however the APC decreased from 1.74 to 0.68 from 2003-2006 to 2006-2019. It would be worth mentioning that in the discussion and whether the authors have any explanation for this.\nAdditionally, as also reported in other SEER studies, the incidence for Blacks is higher than Whites. However, the increase in incidence in Blacks throughout the years has been non-significant in all 3 time-points based on the APC reported here; whereas, this the APC was statistically significant for Whites during 2006-2019. There is a study published in Gastroenterolgy in 2022 using similar database looking at racial disparity which perhaps worth looking at since specifically discussed this issue (info added in citation).\nAnother aspect that should have mentioned in the discussion is the incidence based on stage which shows localized disease is the only stage that is significantly increasing in incidence in the last time point (2010-2019) where as the APC for other 2 stages are statistically not different than 0. This would open the discussion around potential etiologies such as perhaps early detection.\nSimilar pattern is also visible with incidence-based mortality with significant APC only in Whites throughout all 3 time intervals. Does that mean mortality is increasing with faster pace in Whites than other racial groups?\nAnd finally, in Figures 2, the first graph is pancreatic cancer incidence by sex which mistakenly written race.\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? Partly",
"responses": []
}
] | 1
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https://f1000research.com/articles/12-15
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https://f1000research.com/articles/12-13/v1
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05 Jan 23
|
{
"type": "Research Article",
"title": "Genetic mapping and pedigree analysis of non-syndromic congenital deafness in Surabaya, Indonesia",
"authors": [
"Nyilo Purnami",
"Hamam Kusumagani",
"Puguh Setyo Nugroho",
"Zakiyatul Faizah",
"In Seok Moon",
"Hamam Kusumagani",
"Puguh Setyo Nugroho",
"Zakiyatul Faizah",
"In Seok Moon"
],
"abstract": "Background: Genetic mutations cause 50-75% of congenital deafness. Congenital deafness is a disease divided into syndromic and non-syndromic types. Type B special schools facilitate children with deafness in Surabaya. This study aimed to identify a mutation gene that causes non-syndromic hearing impairment in school students. Methods: This research was an analytic observational study using a cross-sectional method applying simple random sampling of non-syndromic deaf children. The applied tests were pure tone audiometry, polymerase chain reaction (PCR), and hybridization tests. Subjects were evaluated on the pedigree route and then analyzed. Results: A total of 138 participants with non-syndromic hearing loss were enrolled in this study and 49 patients met the inclusion criteria. The subject’s average age was 16.16 years, with more male subjects than female. The hybridization results obtained six genetic mutations, one subject with mtDNA 1555, one subject with GJB2-299, two subjects with SLC26A4-IVS(7)2, and 3 subjects with unknown mutations. In pedigree analysis, the same genetic mutation was found in two generations of subjects with mtDNA 1555 and GJB2-299 mutations, and mutations of more than one genetic type were found in two generations of subjects with the SLC26A4-IVS(7)2 mutation. Conclusions: This study obtained six genetic mutations in non-syndromic congenital deaf children. Non-syndromic congenital deafness in Indonesia was found to have genetic mutations as a health risk. The risk was heredity with diverse inheritance patterns.",
"keywords": [
"congenital deafness",
"non-syndromic type",
"school",
"genetic mutation",
"pure tone audiometry",
"hybridization test"
],
"content": "Introduction\n\nHearing loss is a health problem affecting about 6-8% of the population in developing countries and is partly a congenital disability.1 Congenital hearing loss (CHL) is one of the most common disorders in humans, with an incidence of 1-2 out of 1000 newborns.2 Congenital hearing loss can be caused by genetic or environmental factors or the interaction of these two. Genetic factors play about 50-75% as the cause of hearing loss. 30% of congenital hearing loss is syndromic (SHL) with abnormalities in other organ systems, and 70% is non-syndromic. Approximately 600 SHL-related syndromes have been identified, including Usher, Pandred, Stickler, Branchio-to-renal, Down's syndromes, et cetera.2 Based on data from the hereditary hearing loss homepage, 132 mutations genes associated with sensorineural hearing loss (SNHL): 51 autosomal dominant (DFNA), 77 autosomal recessive (DFNB), and 5 X-chromosome (DFNX).2,3\n\nHereditary hearing loss is classified into syndromic and non-syndromic types. The syndromic type accounts for 30% of genetic deafness.4,5 According to World Health Organization (WHO) data, there are 466 million people in the world experiencing a hearing loss (6.1% of the total population), of which 34 million are found in children and 432 million in adults.6 In 2018, 2.6% of the Indonesian population experienced hearing loss. The population with hearing loss in Indonesia is distributed in several age groups, most of which are above 75 years, with a prevalence of 36.6%.7 Congenital hearing loss related to genetic factors is found in two forms, namely: syndromic hearing loss and non-syndromic hearing loss.3 Non-syndromic hearing loss is a hearing loss that is not associated with other physical disorders. Non-syndromic hearing loss can be inherited in autosomal recessive, autosomal dominant, X-chromosome-associated, and mitochondrial inheritance. Non-syndromic hearing loss causes prelingual hearing loss in children with sensorineural types ranging from severe hearing loss to total deafness.4,5\n\nMore than 60 genes and some proteins are involved in the pathogenesis of SNHL. To date, mutations in four connexin genes, including Gap Junction Beta 2 (GJB2)/Connexin 26 (Cx26), GJB3 (Cx31), GJB4 (Cx30.3), and GJB6 (Cx30), have been associated with SNHL.4 Research in Surabaya in 2019 reported that changes or variations in the nucleotide G to A at sequence 8473 in the GJB2 gene, changing the encoded amino acid (valine to leucine) were found in patients with Hereditary Hearing Loss in Deaf School type B Surabaya. This was assumed to be related to the occurrence of congenital deafness.6\n\nGenetically inherited hearing loss affects various molecular processes, including gene mutations that interfere with the function of transcription factors, potassium and chloride channels, connexins, and stereocilia. With genetic testing, the identification of mutations can produce an accurate prognosis for deafness. Genetic information can help predict whether the hearing loss will persist or worsen, and determine the type of damage in the hearing system. Furthermore, knowing the degree of damage to the inner ear could help to determine whether the patients need hearing aid or cochlear implant surgery to improve their hearing.7,8\n\nWHO held a multi-nation consultative meeting in Colombo in 2021 and recommended every stakeholder in various countries, especially in the Southeast Asian region to focus on preventive and early detection efforts related to ear health. This meeting was the basis for the Sound Hearing 2030 program to improve the quality of life in the Asian region by developing a right to better hearing program. With this program, it is expected that the prevalence of hearing loss will be reduced approximately 90% by 2030.9\n\nNon-syndromic hearing loss is a problem because there is lack of knowledge of the cause. In contrast, in the syndromic type, the pathophysiology of typical symptoms could be explored. This study aimed to find out the causes of non-syndromic type congenital deafness through a genetic approach; to detect the presence or absence of genetic mutations that caused deafness; to identify specific mutations, and to ascertain how the pattern of inheritance of these mutations.\n\n\nMethods\n\nThis study was an analytic observational study using a cross-sectional method. This research was done from February 2021 to December 2021 in Karya Mulya type B special school for the deaf in Surabaya, the capital of East Java province, Indonesia. The study design was an analytic observational study using a cross-sectional method. We (with teachers) invited and explained the research to parents of students by the letter with consent form (see Extended data20). Parents and students who wish to participate sign a consent form. There were 50 students have agreed to participate, out of 138 total population. With simple random sampling, we took 49 students as the sample for the study. Inclusion criteria were the students who attended school, cooperative student, has no history of trauma, viral or bacterial infection related to hearing impairment, has parental consent to participate in the study, and has normal ear anatomy. Students who did not meet the inclusion criteria were excluded.\n\nFirst, we interviewed students and parents accompanied by teachers so that we could communicate and obtain the clinical history of students at Karya Mulya special school. It spent 5 active days to finished 49 students. The clinical history of each subject was explored to ensure that the hearing loss was not a result of acquired environmental factors like infection, trauma, acoustic trauma or ototoxic drugs. All subjects were investigated through physical and otological examinations by an ENT specialist to find the presence of other symptoms that could point to a syndromal type of hearing loss, including diabetes, vision problems, neurological disorders, and skin disorders. Subjects with possible syndromal or acquired hearing loss were excluded from the study. Using standard procedures, the DNA from all patients were extracted from the patient’s blood sample.\n\nThe data were obtained from medical record data of the patient, audiometric examinations, blood analysis using polymerase chain reaction (PCR) and hybridization, then processed to determine the type of genetic mutation associated with deafness. Pure tone audiometry was performed using an Eartech Resonance r17a-BC Portable Touchscreen Audiometer by ENT doctor, in a room with a noise level of not more than 40 decibels for about 5 to 10 minutes to determine the residual hearing threshold.\n\nThe workflow in this research was implemented in three steps: 1) Human DNA Extraction from sample, 2) PCR Amplification, and 3) Hybridization. The Hearing Loss Susceptibility GenoArray Diagnostic Kit contains sufficient reagents for 30 tests.\n\nHuman DNA extraction sample can be taken from whole blood, dried blood spot or buccal swab. In our study, 6 ml of patient’s blood. was extracted by the nurse and stored at the cool box. The collected sample was sent for test immediately to the Prodia laboratory for hybridization process. The blood sample can be stored at the cooler with temp 4oC for less than 24 hours or -20°C for less than one month to prevent denaturation.\n\nDNA extraction was executed according to the Hybribio DNA Prep (HBDP) kit instructions book in August 2017 on the revision date. DNA Prep Kit is designed for extracting DNA from whole blood (with anticoagulant) for clinical diagnosis or research purposes. Principle of the test, DNA was released from whole blood using silica spin column DNA purification. This kit contents of solution L (<10%GuSCN, <10%Tris), solution P (<1% KH2PO4, <1%NaCl), solution W1 (<10% GuSCN, <1% EDTA), solution W2 (sterilized deionized water), TE Elutin buffer (<1% Tris, <1% EDTA) and protease K (<2% protease K). Test procedures:\n\n1. Add 200μl anticoagulant blood sample into a 1.5ml-eppendorf tube. If the sample is less than 200μl, add Solution P into the sample to mark it up to 200μl.\n\n2. Add 20μl Protease K into the blood sample, and shake to mix well\n\n3. Add 200μl of Solution L, vortex the sample (we use Thermolyne Maxi Mix II Vortexer), then place it in a 56°C water bath or electronic hot bath for 15-20min with a digital block heater.\n\n4. Add 200μl of ethanol and mix well. Insert one 2ml-centrifugal filter tube in one collection tube, transfer the sample into the 2ml-centrifugal high filter tube, centrifuge at 1000rpm for 1 min, and discharged the flow-through.\n\n5. Add 500μl of Solution W1 to the tube and centrifuge at 1000rpm for 1min, discard the flow-through\n\n6. Add 500μl of Solution W2 to the tube and centrifuge at 1000rpm for 1min, discard the flow-through\n\n7. Repeat step 6\n\n8. Reattach the centrifugal high filter tube back into the collection tube, and centrifuge at 12000rpm for 3 min. Discard the flow-through and the collection tube.\n\n9. Transfer the centrifugal tube into a new 1.5ml-Eppendorf spin tube and allow the tube to sit for 1-2min with the cap opened. Add 80μl of TE Elution Buffer, allow the tube to sit for 5 min, and centrifuge at 12000rpm for 2min to elute the DNA.\n\nTo make sure the DNA extraction was pure, we performed electrophoresis. We used agarose gel electrophoresis with HE-PLUS by Hoefer and GelDoc XR by BIO-RAD to interpret it.\n\nThe principle of Hearing Loss Susceptibility GenoArray Diagnostic Kit was using polymerase chain reaction (PCR) to amplify extracted DNA from PB (Peripheral Blood). PCR is a laboratory technique used to make many copies of a particular region of DNA (until millions or billions). The goal of PCR is to make enough of the target DNA region so that it can be analyzed.\n\nPCR amplification area must be separated from PCR reagent preparation workstation. The reagent has 3 labeled (PCR mix, DNA Taq, and control negative (-)). PCR mix devided into 2 types (PCR mix A and B) with the same composition; Tris-HCl buffer, MgCl2, (<3% dATP, dCTP, dGTP, dTTP), (<3% synthetic oligonucleotide primers) and the same volume (825μl). The DNA Taq contained 30μl of DNA polymerase (5U/μl) and stabilizer. The negative control contained 100μl of DNAse-free distilled water.\n\nPrepare each PCR Amplification Reagent tube according to the following pipetting scheme (total volume/reaction: 28 μl). Take a blood sample from the -20°C storage, and the DNA was extracted using PCR standard procedure, then take 2μl of the extracted DNA sample and placed as group1 and group 2. DNA was extracted using standard PCR procedures. Amplification of the DNA segment and denaturation was made with Bio-Rad CFX96 Touch Real-Time PCR Detection System, USA. We used 4°C/sec ramp rate for the thermocycler and re-examined the DNA amplification with agarose gel electrophoresis.\n\nThe hybridization kit used in the study was Hearing Loss Susceptibility GenoArray Diagnostic Kit (ref HB_HLS GA) by Hybribio limited, Sheung Wan, Hong Kong) to analyze genetic mutation. Thirteen mutations in four genes (GJB2, GJB3, SLC26A4 and 12S rRNA) are evaluated simultaneously. By using polymerase chain reaction (PCR) to amplify extracted DNA from PB (Peripheral Blood), amplified DNA amplicons are then hybridized with specific probes located inside the “HybriMem box” under hybribio patented “flow-through hybridization” technology followed by colorimetric result obtained using enzyme immunoassay method without using primer as in DNA sequencing (Figure 1).\n\n(A) HybriMem Box. The wells detect specific gene mutation. (B) DNA HybriMax HHM-3 device, containing 15 HybriMem Box.\n\nThe hybridization reagents consist of hybridization solution, blocking solution, enzyme conjugate, solution A, washing solution, NBT/BCIP, hybridmem box. The composition of hybridization solution were 120ml 2X SSC and 0.1% SDS. The composition of blocking solution was 30ml TBS with <0.1% detergent. The composition of enzyme conjugate were 15ml <0.0003% Streptavidin-Alkaline, Phosphatase Conjugate, and Stabilizer. The composition of solution A was 100ml Tris-HCl buffer with 0.05% sodium azide. The composition of washing solution were 96ml 0.5X SSC and <0.1% SDS Solution. The composition of NBT/BCIP was 15ml 1-5% (w/w) NBT/BCIP. The composition of hybrimem box was 30 Nylon Membrane coated with specific DNA probes.\n\nThere were 3 steps in hybridization process. First step was running at 45°C from (number 1-11), second step (number 12-20) at 25°C, and third step (number 21-28) at 36°C. The steps were:\n\n1. Set HybriMax temperature at 45°C. (Step 1-11 running at 45°C)\n\n2. When the temperature of HybriMax reaches 45°C, add 0.8ml of 45°C pre-warmed Hybridization Solution into the sample well where “HybriMem” is located for 2 min.\n\n3. Pump away all the solvent, then pump off.\n\n4. Add 0.8ml of 45°C pre-warmed Hybridization Solution into the same wells.\n\n5. Add group 1 and group 2 denatured DNA amplified samples from PCR tubes into one well, pipetting 2-3 times to mix the solution carefully.\n\n6. Close the lid.\n\n7. Incubate for 20 min. (Hybridization)\n\n8. Pump away all the solvent, then pump off.\n\n9. Add 0.8ml of 45°C washing Solution. (Washing Step)\n\n10. Repeat Steps 8-9 four times more.\n\n11. Pump off.\n\n12. Set HybriMax temperature at 25°C.\n\n13. When the temperature of HybriMax reaches around 30°C, add 0.5ml of Blocking Solution into the well. (Temperature is going down from 45°C to 25°C)\n\n14. Pump away all the solvent, then pump it off.\n\n15. Again, add 0.5ml of Blocking Solution and Incubate for 5 min; pump away all the solvent, then pump off.\n\n16. When the temperature of HybriMax reaches 25°C, add 0.5ml of Enzyme Conjugate and Incubate for 5 min.\n\n17. Pump away all the solvent then pump off.\n\n18. Add 0.8ml of Solution A. (Washing Step)\n\n19. Repeat Step 17-18 four times more.\n\n20. Pump off.\n\n21. Set HybriMax temperature at 36°C.\n\n22. When the temperature of HybriMax reaches 36°C, add 0.5ml of NBT/BCIP Solution (brown bottle) and Incubate for 5 min. Close the lid. (Coloring)\n\n23. Pump away all the solvent.\n\n24. Keep the pump on.\n\n25. Add 0.8ml of Hybridization Solution. (Washing Step)\n\n26. Repeat Step 25 three times more.\n\n27. Add 1.0ml of Distilled Water. (Rinsing Step)\n\n28. Pump off\n\n29. Remove the fixing cover and all accessories. Using forceps to take out the membranes and dry them on absorbent paper.\n\n30. Interpret the results by color visualization observed on membrane.\n\nStatistical analysis was carried out using descriptive techniques in Microsoft Excel and then interpreted in tabular form, using the SPSS Statistics 23 software.\n\nEthical clearance was obtained from the Health Research Ethical Committee of Medical Faculty Universitas Airlangga, Surabaya, Indonesia (approval number 9/EC/KEPK/FKUA/2021) in accordance with 7 (seven) WHO 2011 standards in October 2020. Written informed consent was obtained from both all subjects included in this study, as well as parents and teachers, after a thorough explanation of the examinations that would take in the study.\n\n\nResults\n\nThe characteristics of 49 subjects were as follows: the percentage of male and female participants was 51% and 49%. There were no subject with age≤10 years, nor >30 years, the highest number of subjects was in age range 11-20 years (91.84%), with the highest education level was senior high school 28 subjects (57.14%) (Table 1).18\n\nThe results of the audiogram examination, showed the type of deafness was sensorineural in all subjects, with the highest number was profound hearing loss (>90dB) in 46 subjects (93.88%), and the least number was severe hearing loss (71-90dB) in right ears 3 subjects, and left ears 3 subjects (6.12%) (Table 2)\n\nThe results of the hybridization examination in Table 3 showed that genetic mutations occurred in 6 subjects (12.25%) with criteria for genetic mutations of the GJB2 gene 1 subject (2.04%), PDS gene 1 subject (2.04%), mtDNA gene 1 subject (2.04%), and in 3 subjects (6.13%) unknown (genetic mutation was found, but the type was unknown).19 The pattern of inheritance of the genetic mutation occurred in autosomal recessive 1 subject (2.04%), sex-linked 1 subject (2.04%), and mitochondrial 1 subject (2.04%).\n\nGJB2 family\n\nThe genetic mutation of the GJB2 gene was indicated by the results of the hybridization of subject 49 (Figure 1). It could be seen that in column 299M of the hybridization panel appeared a dot, so the form was read as genetic mutation of GJB2-299M. The pedigree pattern in the first family generation from the medical record was unknown, as they could not be reached, the second generation (patient's parents) showed a mutation in the father, but no phenotype appeared (unimpaired hearing). While the mother was non-mutated, there was no mutation. The same mutation was inherited in both boys, but only the first child showed a non-syndromic congenital deafness phenotype, and the second child had normal hearing.\n\nmtDNA family\n\nIn patients with hybridization results, the mtDNA genetic mutation was shown through subject 55, characterized by the disappearance of a dot in the 1555N column and the appearance of a dot in 1555M (Figure 1). The pedigree pattern of samples with mtDNA genetic mutations revealed that the first generation was known to have deafness in number 1 and unimpaired hearing in numbers 2, 3, and 4. The second generation of fathers had deaf sons and non-deaf daughters. In the second generation of mothers, there was no deafness. The third generation had offspring from the father and mother with a deaf phenotype. Both male and female children had the same phenotype and the same genetic mutation as the mother, while there was no mutation from the father’s side.\n\nSLC26A4 family\n\nPatients with the SLC26A4 genetic mutation were shown by hybridization results in the presence of dots in the IVS-M and IVS-N columns in samples 51 and 52 (Figure 1). In the pedigree pattern, mutations were found in the first and second generations, namely in the patient's grandmother and mother, but did not show a deafness phenotype (unimpaired hearing). Depicted in this pedigree pattern, in a non-deaf grandmother was detected more than one genetic mutation, passed on to a non-deaf daughter (patient's mother) who had a similar mutation, but when passed down to the males of third generation, the deafness phenotype appeared with heterogeneous IVS-M and IVS-N mutations.\n\n\nDiscussion\n\nNon-syndromic congenital deafness has 70% of the features that often appears in congenital hearing loss. The most common forms of inheritance in NSHL are autosomal recessive 77%, followed by autosomal dominant 22%, X-linked 1%, and mitochondrial <1%. 8 Genetic mutations that cause NSHL vary between different populations and ethnicities. The most common mutation is GJB 2 which is encoded by the protein connexin 26. The population in Saudi Arabia has higher OTOF gene mutations than the GJB2 gene. Mutations of GJB2, GJB3, SLC26A4, and mitochondrial 12SRNA were common causes of NSHL found in China.7,10\n\nA similar study in Surabaya, Indonesia, showed that there were more females than males with hearing loss, different in Iraq where there were more males than females. Gender has no significant relationship with the occurrence of deafness.11\n\nThe results of the audiogram examination, showed that the type of deafness was sensorineural in all subjects, with the highest number was profound hearing loss (>90dB) in 46 subjects (93.88%). In a research profile of 430 patients with congenital deafness in America, the highest degree of the hearing was very severe (>90dB) in 133 samples, with no lateralization (symmetrical) in 311 samples (Raymond, 2019).12 Hearing loss may manifest in the ears as bilateral or unilateral and symmetrical or asymmetrical. Genetic deafness was mostly found as bilateral (44%), asymmetrical (22%) and unilateral (2%). There was a negative correlation between age and deafness after environmental factors were excluded in 1119 patients; congenital onset occurred in 45%, childhood 30%, and adulthood 28%. A study of 200 samples in the Netherlands stated that deafness could be found 50% as congenital, 38% in the first decade, and 20% in the second decade of life.2\n\nThe results of the hybridization examination in Table 1 showed that genetic mutations occurred in 6 subjects (12.25%), with criteria for genetic mutations of the GJB2 gene 1 subject (2.04%), PDS gene 1 subject (2.04%), mtDNA gene 1 subject (2.04%), and 3 subjects (6.13%), unknown (genetic mutation was found but the type was not known). Research in Bosnia and Herzegovina described that in the results of exome sequencing of patients with non-syndromic deafness, 68% showed negative results, 18% positive mutations, while in syndromic deafness it was more genetic mutations were found.13 Following this study, genetic examinations for non-syndromic deafness did not always reveal mutations. In this study, due to limitations in the examination, only a few genes could be detected.\n\nThe pattern of inheritance of genetic mutations in this study occurred in autosomal recessive one subject (2.04%), sex-linked one subject (2.04%), and mitochondrial one subject (2.04%). Non-syndromic hearing loss (NSHL) was inherited in an autosomal recessive manner (75-80%), autosomal dominant (20-25%), and 1-2% X-chromosome and mitochondria-linked. After aging, the prevalence of autosomal dominant inheritance and mitochondrial inheritance increases while autosomal recessive inheritance decreases.7,14 Autosomal dominant is described as a child whose mother has a no mutations gene and a father who mutates the dominant gene, can inherit a 50% chance of being deaf. Only one copy of the inherited mutation gene can cause deafness in a child. So in every pregnancy, there is a 50% chance that the child will be deaf. Autosomal recessive is when the chromosomes of the father and mother have a recessive mutation in the same gene and are inherited in 50% of their offspring, but it takes two mutated genes to produce a deafness phenotype. In X-linked inheritance, boys have a higher chance of deafness. Mitochondrial inheritance is only inherited by eggs from the mother, and all offspring will be deaf.15\n\nThe results of hybridization of research and family samples obtained one sample of IVS-M/IVS-N mutation; one sample of 299M/299N mutation; one sample of the 1555M mutation; one sample of mutations 235M/235N, 7445M/7445N, and IVS-M/IVS-N; one sample of mutations 235M/235N, 7445M/7445N, 538M/538N, and IVS-M/IVS-N. The interpretation of the results of this hybridization was, if an IVS-M/IVS-N mutation was found, it was defined as a SLC26A4-IVS(7)2 mutation; mutation 299M/299N defined as GJB2-299; the 1555M mutation was defined as mtDNA1555; mutation 235M/235N defined as heterozygous GJB2-235 mutation; mutations 7445M/7445N defined as mtDNA7445 mutations, mutations 538M/538N defined as mutations GJB3-538. The family of patients with mtDNA 1555 (1555M homozygous) had a deaf father and mother, but only the mother had the 1555M mitochondrial mutation. The result of inheritance in their offspring was deafness in all children. This was related to the reference where mitochondrial inheritance can only be inherited by egg cells from the mother, which caused all of the offspring to be deaf.15,16\n\nThe hybridization kit used by researchers had limitations in assessing the genetic mutations that occured. The Hearing Loss Susceptibility GenoArray Diagnostic Kit is designed for rapid screening with accurate results through dot mutations known to be associated with hereditary hearing loss. Thirteen mutations in four genes (GJB2, GJB3, SLC26A4 and 12S rRNA) were evaluated simultaneously. In this study, Knowledge of mutations could help identify hearing loss at birth and could advise avoiding taking certain types of antibiotics that are associated with deafness in children who carry the gene mutation.17 Researchers took hybribio as a genetic mutation screening method because the GJB2, GJB3, mitochondrial and SLC26A4 genes are genetic mutations that often appear in Asian populations.17\n\nIn the pedigree pattern, the SLC26A4 family (IVS-M/IVS-N) had its own pattern, where the grandmother had 4 genetic mutations (235M/235N, 7445M/7445N, 538M/538N, IVS-M/IVS-N), the mother had 3 genetic mutations (235M/235N, 7445M/7445N, IVS-M/IVS-N), and the child had only 1 genetic mutation. This was interesting because the inherited mutation was only 1 gene while the other genes were missing (235M/235N, 7445M/7445N, 538M/538N). The most common genes in cases of autosomal recessive hearing loss in order of highest frequency are the GJB2, SLC26A4, and MYO15A genes.7\n\nIn the GJB2 family pattern (299M/299N heterozygous) a father who carried the genetic mutation but was not deaf with a mother not found the mutation gene, had deaf and normal sons. This pattern was an autosomal recessive genetic mutation. Under the reference that in cases of non-syndromic hearing loss, the most common mutation occurred in GJB2.7\n\n\nConclusion\n\nNon-syndromic congenital deafness in Surabaya was caused by genetic mutations GJB2, SLC26A4 and mtDNA 1555, which were passed down through families with diverse inheritance patterns. Genetic mutation is one of the causes of non-syndromic congenital deafness. Hybridization examination is a limited screening method to determine promptly the presence of genetic mutations that caused deafness in GJB2, GJB3, mtDNA, and SLC26A4 mutations that often occur in Asia. Genetic counselling is needed to predict the risk of deafness that will be passed down, prepare genetic screening methods, and be able to determine the prognosis of the therapy that will be carried out. Further examination with next generation sequencing method on all samples is needed to find more genetic mutations that cause non-syndromic deafness.",
"appendix": "Data availability\n\nFigshare: Genetic Mapping and Pedigree Analysis of Non-syndromic Congenital Deafness in Surabaya, Indonesia. https://doi.org/10.6084/m9.figshare.21739010.v1. 18\n\nFighsare: Result of PCR and hybridization. https://doi.org/10.6084/m9.figshare.21749282.v1. 19\n\nFigshare: Inform Concent of genetic patient. https://doi.org/10.6084/m9.figshare.21679319. 20\n\nThis project contains the following extended data:\n\n- Consent form\n\n- Data collection sheet\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).\n\n\nAcknowledgments\n\nThe authors would like to express their gratitude to Karya Mulia Deaf School Surabaya and Prodia Clinical Laboratory and to Simlitabmas LPPM Universitas Airlangga. The authors are also very grateful to the Department of Otorhinolaryngology Faculty of Medicine, Universitas Airlangga.\n\n\nReferences\n\nPurnami N: The modified whispered test for screening of hearing impairment in children at the elementary school. J. Phys. Conf. Ser. 2018; 1: 1–5.\n\nVona B, Doll J, Hofrichter MAH, et al.: Non-syndromic hearing loss: Clinical and diagnostic challenges. Med. Genet. 2020; 32(2): 117–129.\n\nCamp VG, Smith RJH: The Hereditary hearing loss home page.2019. Accesed Dec 21, 2021.Reference Source\n\nMorton C: Genetics, genomics, and discovery in the auditory system. Hum. Mol. Genet. 2002; 11: 1229–1240.\n\nToriello HV, Reardon W, Gorlin RJ: Hereditary hearing loss and its syndromes. New York:Oxford University Press;2004.\n\nPurnami N, Prabowo GI, Wungu CD, et al.: Detection of single-nucleotide polymorphism Gap junction protein Beta-2 genes in deaf schoolchildren of Javanese population in Surabaya, Indonesia. Indian J. Otolaryngol. 2019; 25: 6–10.\n\nVenkatesh MD, Moorchung N, Puri B: Genetics of non syndromic hearing loss. Medical Journal Armed Forces India. 2015; 71(4): 363–368.\n\nMishra S, Pandey H, Srivastava P, et al.: Connexin 26 (GJB2) mutations Associated with Non-Syndromic Hearing Loss (NSHL). Indian J. Pediatr. 2018; 85(12): 1061–1066.\n\nChadha S, Cieza A, Krug E: Global hearing health: future directions. Bull. World Health Organ. 2018; 96(3): 146.\n\nChen S, Liang Z, Chen B, et al.: The prevalence of deafness-associated mutations in neonates: A meta-analysis of clinical trials. Int. J. Pediatr. Otorhinolaryngol. 2019; 121(17): 99–108.\n\nAl-janabi AM, Ahmmed HS, Al-khafaji SM: Connexin 26 (GJB2) Gene mutations linked with autosomal recessive nonsyndromic sensorineural hearing loss in Iraqi population.2021; 26: 1–11.\n\nRaymond M, Walker E, Dave I, et al.: Genetic testing for congenital nonsyndromic sensorineural hearing loss. Int. J. Pediatr. Otorhinolaryngol. 2019; 124(May 2019): 68–75.\n\nLikar T, Hasanhodžić M, Teran N, et al.: Diagnostic outcomes of exome sequencing in patients with syndromic or nonsyndromic hearing loss. PLoS One. 2018; 13(1): 1–14.\n\nWalls WD, Moteki H, Thomas TR, et al.: A comparative analysis of genetic hearing loss phenotypes in European/American and Japanese populations. Hum. Genet. 2020; 139(10): 1315–1323.\n\nZahara D, Bashiruddin J, Tann G, et al.: Gap junction beta 2 gene mutation in indonesian patients with non syndromic congenital hearing loss. Int. J. Pharmtech Res. 2015; 8(9): 69–76\n\nRehm LH, Williamson ER, Kenna AM, et al.: Understanding the genetics of deafness: A guide for patients and families. Cambridge:Harvard Medical School Center for Hereditary Deafness;2003; 4–10.\n\nUsami S, Nishio S:Nonsyndromic Hearing Loss and Deafness, Mitochondrial. 2004 Oct 22 [Updated 2018 Jun 14]. Adam MP, Everman DB, Mirzaa GM, et al., editors. GeneReviews®. Seattle (WA):University of Washington, Seattle; 1993-2022.Reference Source\n\nPurnami N:Genetic Mapping and Pedigree Analysis of Non-syndromic Congenital Deafness in Surabaya, Indonesia. [Dataset]. figshare. 2022. Publisher Full Text\n\nPurnami N:Result of PCR and hybridisation. figshare. [Dataset]. Figure. 2022. Publisher Full Text\n\nPurnami N:Inform Concent of genetic patient. [Dataset]. figshare. 2022. Publisher Full Text"
}
|
[
{
"id": "173018",
"date": "26 May 2023",
"name": "Arunachalam Iyer",
"expertise": [
"Reviewer Expertise Otology",
"middle ear pathology",
"hearing reconstruction",
"bone anchored and middle ear implants",
"cholesteatoma"
],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 addresses an important aspect of SNHL in non-syndromic children. This will help to understand the pathology and counsel the parents about the prognosis and risks for the siblings. While it is a fairly well-written paper, it could be made even better by addressing some minor issues as follows:\nIn the Introduction: correct the spelling of \"Pendred\" syndrome\n\nIn the discussion section, the following paragraph needs correction. Autosomal dominant can come from either parent, not only the father. This correction is needed to clarify the condition. \"Autosomal dominant is described as a child whose mother has a no mutations gene and a father who mutates the dominant gene, can inherit a 50% chance of being deaf. Only one copy of the inherited mutation gene can cause deafness in a child. So in every pregnancy, there is a 50% chance that the child will be deaf. Autosomal recessive is when the chromosomes of the father and mother have a recessive mutation in the same gene and are inherited in 50% of their offspring, but it takes two mutated genes to produce a deafness phenotype. In X-linked inheritance, boys have a higher chance of deafness. Mitochondrial inheritance is only inherited by eggs from the mother, and all offspring will be deaf.15\"\n\nIn the methods section, it appears that the majority of children in the school were excluded from the study! Is it only because of the parental consent issues? If so, was a more detailed explanation given to parents to encourage to recruit?\n\nThere are minor grammatical corrections needed to many areas of the paper, this can be achieved easily by using a grammar editor. This will make the paper more easily readable and enjoyable in my opinion\n\nIs the work 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": "9718",
"date": "29 Nov 2023",
"name": "Nyilo Purnami",
"role": "Author Response",
"response": "Thank you for taking the time to review our paper, allow me to provide feedback on your revisions and questions: 1. Yes, I will do it. 2. Thanks for your suggestion, I will revise according to your suggestion. “Autosomal dominant can come from either parent, not only the father\" 3. Study design and participants This study was an analytic observational study using a cross-sectional method. This research was done from February 2021 to December 2021 in Karya Mulya type B special school for the deaf in Surabaya, the capital of East Java province, Indonesia. The study design was an analytic observational study using a cross-sectional method. We (with teachers) invited and explained the research to parents of students by the letter with consent form (see Extended data20). Parents and students who wish to participate sign a consent form after we explained the purpose, research and examination steps, complication, and guaranteed treatment if there's complication. There were 50 students who meet the inclusion criteria that agreed to participate, out of 138 total population. With simple random sampling, we took 49 students as the sample for the study. Inclusion criteria were the students who attended school, cooperative student, has no history of trauma, viral or bacterial infection related to hearing impairment, has parental consent to participate in the study, and has normal ear anatomy. The majority of children in the school were exclude because of parental issues who's still worried when we took the blood for examination, has a history of ear trauma, infection and ototoxic medication. 4. Yes, I will do revisions minor grammatical corrections. Thank you for taking the time to review our paper, allow me to provide feedback on your revisions and questions"
}
]
},
{
"id": "219577",
"date": "22 Jan 2024",
"name": "Elvis Twumasi Aboagye",
"expertise": [
"Reviewer Expertise Human Genetics"
],
"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\n1. Random sampling method used for participants selection not clear - how the 49 participants were selected 50 agreed to participate / simple random sampling method to select 49 participants out of the 50...? Clarity required. 2. Repeated statement needs to be revised or remove. 3. Standard presentation of genes (italics) and protein symbols. GJB2 coding sequence size (exon 2) and 8473 position for the identified variant.? Not clear and use standard presentation of reporting variants for easy comprehension and understanding. 4. The study may not lead to identifying causal variant...revise strong statement that suggest and establish causality. 5.SOP presented not relevant state the kits used for the DNA extractions and quality controls performed to ensure DNA quality same applies to the hybridization. 6. Not clear if only the probands were sampled and investigated, and pedigree to demonstrate family history or no history not shown...only mentioned for the variants and pedigree not presented. 7. The variants identified were not validated after the hybridization assay...? 8. Not clear what authors mean by unknown variant should clarify. what gene, genomic location, and details... not sure is novel to their population or deafness associated variants. 9. Article needs extensive revision for consideration.\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": "227994",
"date": "25 Jan 2024",
"name": "Adebolajo Adeyemo",
"expertise": [
"Reviewer Expertise Otolaryngology"
],
"suggestion": "Not Approved",
"report": "Not Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis is an analytical observational study of children with non-syndromic deafness in Indonesia. The authors made great effort to recruit children from the school of the Deaf.\nThe manuscript however requires a lot of modifications to make it of a good quality.\nThe abstract background should be re-written to accurately depict the goal of the study. Abstract results: 139 students were not enrolled in the study. That was the total number of students in the school, out which 50 students consented and 49 enrolled for the study. Abstract conclusion:How is genetic mutation an health risk of NSSNHL? Study design: Out of 50 consented students, how is it possible to randomly select 49 students. Especially when the abstract showed that the 49 students where the ones that met the inclusion criteria. Measurements: The detailed guide for the DNA extraction, PCR amplification and hybridization is unnecessary. Appropriate references to direct the user to the source materials is sufficient. Discussion: The opening paragraphs were literature review that ought to be sited in the Background section of the manuscript. Discussion: There are instances of dual referencing style: in 311 samples (Raymond, 2019).12 Discussion: There are multiple grammatical errors. The manuscript will benefit from language editing. Discussion: I believe the description of Autosomal recessive and dominance is not appropriate in this context.\n\nI believe there is need for an overhaul of the manuscript to improve the quality and enable its contribution to the body of knowledge.\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": []
}
] | 1
|
https://f1000research.com/articles/12-13
|
https://f1000research.com/articles/12-9/v1
|
04 Jan 23
|
{
"type": "Opinion Article",
"title": "Judicial breakfast as an external factor in judicial decision making in courts",
"authors": [
"Manotar Tampubolon",
"Tomson Situmeang",
"Paltiada Saragih",
"Tomson Situmeang",
"Paltiada Saragih"
],
"abstract": "The objective of this article is to establish whether the judges depend on relevant facts, judicial rules, and the law when making their judgments or they use extraneous factors such as what a judge eats, personal ideology, beliefs, or the cultural and political environment. The discourse between the two sides is incomplete without exploring the grand theories: formalism and realism. The antimony between the two theories resulted in theoretical analysis and empirical research. The realism challenged the existing logical reasoning and legal rules that judges use in making their judgment as they contend that judges applying rules and law in their decision-making process are irrational and mechanical. Formalism insists on using the judicial rules and the law in making decisions as opposed to extraneous factors, which realists contend should be the basis for decision making with laws and rules only to support the findings. The continental legal theory holds that legal realism is a hard-nosed, down-to-earth, and practical school of thought that is opposed to mechanical and scientific theories. The scholarly analysis of the judicial decision-making process brings into focus the conduct of judicial officers and whether they base their reasoning on extrajudicial issues. However, the discussion should avoid denigrating into an attack on the personality of judges as it undermines the rule of law.",
"keywords": [
"court",
"extraneous factors",
"formalism and realism",
"legal realism",
"judicial decision making",
"judicial officers",
"judicial rules and laws",
"judicial officers."
],
"content": "Introduction\n\nThe adage that judicial decisions by judges depend on what they ate in their breakfast presents a discourse on whether outcomes of legal cases rely heavily on facts and laws alone. Legal formalism refers to the systematic, logical, and purposeful application of facts and arguments to situations by judicial officers (Aiken & Shalleck, 2016). The alternative claim is that legal realist movements in the 20th century promoted the judicial idea that decisions are grounded in the logical application of the law. It was observed that the life of the law is premised on experience rather than the law. The Supreme Court Judge, Oliver Holmes argued that the balanced application of the law is dependent on the political, psychological, and social factors rather than only legal reasoning and underpinnings (Aletras et al., 2016; Auerhahn et al., 2017; and Bator, 2020). The old trope that judges’ decision-making process is dependent on what they ate presumes that law is subject to imperfections, biases, and foibles that affect human cognitive function. Indeed, people would appreciate it if judges made their ruling based on the written laws and rational decisions; however, the reality is that their decision is influenced by irrelevant factors such as their breakfast and mood.\n\nExisting research shows that making repeated decisions and judgment often depletes the mental resources and executive functions of the individual, which influences their decision-making process (Bonica & Sen, 2021; Boyd, 2016; and Bozorgmehr & Naseri, 2020). Sequential decisions between consumer goods affect the intuitive decisions of humans and lessen the tolerance to pain, which makes them tend to simplify decisions. Social science assumes that the decisions that human beings make are dependent on rationality, are unemotional and logical. However, the neoclassical price theory hypothesizes that consumers and producers are rational actors but in law a reasonable person is a cousin to economic actors (Burns, Dioso-Villa & Rathus, 2017). The dominant decision-making process in the judicial industry is an extension of the rational choice theory where the judicial officers are rational actors that base their actions on previous decisions, facts, constitution, and statutes. The problem with the theory is that it only accounts for a portion of decision-making processes since, from a realist perspective, non-doctrinal considerations were disregarded even though they have an impact on judicial judgments' outcomes. Cognitive scientists and behavioral psychologists have explored how the brain operates, which brings the foundation for examining the actions of judges. Studies show a surprising correlation between emotional and cognitive health and the state of the digestive system, which highlights the importance of judges making a decision when they take breakfast (Chen, 2016). To understand how judicial decision-making is performed, understanding how the mind operates is critical in validifying or refuting the claims that judges make a decision depending on factors other than the law and facts. Establishing whether there are existing choices, competing priorities, and decisions that judges make, as well as their perceptions on issues, is critical.\n\n\nMethods for assessing the decision-making process\n\nThe adage that judges make a decision based on what they consume presents a discourse on factors that judges use when making judgments in courts. The realists contend that the courts are influenced by extralegal factors when making judgments while the formalists contend that judges only rely on facts, laws, and legal rules (Chen, Moskowitz & Shue, 2016). The realists assert that judges do rely on precedence, and are influenced by policy perspectives, and personal feelings, which the formalists disagree on. They argue that judges are not scientists that make a mechanical decision in adherence to rules rather than reality (Chen, 2019). On the other hand, the formalists contend that giving judges free will to make judgments based on their personal opinion, beliefs, and values departs from constitutionalism. To settle the differences, testing for factors affecting the judicial decision-making process requires an experiment. Following DLA theory, the study will seek to establish whether judicial decisions only consider facts, laws, and legal rules or are influenced by extraneous factors.\n\nFor realists, the use of scientific methodology such as experimental research designs is essential in answering the question. Hypothetically, they can conduct an experiment that involves two groups, an experimental group that tests the independent variable and a control group that measures the dependent variable. On the one hand, the experimental group should be made of experienced legal practitioners who have handled many cases. These lawyers are provided with a large sample of cases, an assembling of facts, and asked to predict the outcome of the case. They should use the facts and the number of cases to predict the reasoning of the court. In this group, the participants will not be provided with information such as names and personalities of judges, judicial locale, parties in the case, prevailing cultural and social norms, and emotional appeals that lawyers presented during the hearing of a case. In essence, the participants will be provided with formal legal rules and facts devoid of other extraneous factors such as what the judges ate or how tired they were. On the other hand, the control group will be provided with facts about a case and asked to make predictions about the court reasoning and outcome of the case. They will be denied formal rules and asked to predict the ruling; possibly, by tossing a coin. The control group should be made of a large sample size capable of producing high confidence levels of about 50%.\n\nThe research question for the study would be whether the lawyers could predict the court ruling correctly while using the legal rules and laws to make the predictions compared to the control group. Based on the reasoning of realists, the answer to the question would be largely clear and favorable to their assertion that it is difficult to predict a court decision when only the law and legal rules are presented. Realists argue that scientific theory adopted by formalists is wrong or incomplete because it can only explain events in the past, but cannot predict future events. The realists would agree that it is possible to make predictions when a scientific theory that uses legal rules and law to make judgments is adopted together with policy principles, the personality of a judge, attributes of litigants, judicial ideology, cultural elements during the hearing, and the emotional elements present in the case.\n\nThe findings of the study can be confirmed by contemporary empirical studies. Research conducted by Posner notes that the outcome of a Supreme Court case in the US can be predicted when variables are assessed, which includes no legal doctrine as opposed to a set of experienced constitutional lawyers with facts and an understanding of legal rules. The main objective of the realists is to develop a theory of judging by establishing the factors that determine judicial decision-making. Examining the decision-making process in the judiciary until one can confidently predict judgments is at the center of realists' studies. Though the realists argue against the assertion that law is a legal science, their proposition is best explained using a scientific inquiry.\n\n\nLiterature review\n\nThe constitution allows the judge discretion to make decisions as they are permitted to develop findings of facts, design remedies for violation of the law, interpret the language of the constitution, regulations, and statutes, and grant injunction relief. As well, the judges are allowed to establish whether the government officials in the executive arm have abused their discretion (Cserne, 2020; Dagan, Kreitner & Kricheli-Katz, 2018). However, the reality is that the judges are constrained on how they exercise these powers as they can't act on their desire. First, the judges are constrained by their self-respect as they consider their rulings examined and assessed by many people. When judges make a ruling, they consider how practitioners consider their reasoning, which should be based on solid grounds, facts, and reasonable suppositions (Diamond, 2019). Law practitioners expect judges to make decisions and support them with convincing facts. When they choose to avoid following a principle for them to reach an outcome, it is difficult to argue that one is following the law when making a ruling (Gordon, 2020). Whether one thinks that the judgment is wrong or right, their moral compass and self-respect constrained them into making decisions that are right regardless of their desires.\n\nElliott (2016) adds that the judges are constrained by their colleagues as their decisions are reviewed by the judges in the upper courts. As such, they are forced to make rulings that are convincing, accurate, and adhere to the law. It is integral that they decide to use convincing facts as they have to persuade the upper court that their ruling was correct and reasoning valid. In hearing an appeal, the oversights and errors that a judge in the lower courts makes are often paraded which can be embarrassing. As such, the judges consider the opinions and stances of their colleagues to avoid embarrassment during the hearing of an appeal (Hamer & Edmond, 2016). Although the judges can cut corners with their judgment, their actions often catch up with them as their opinions can be subjected to scrutiny by the Supreme Court. There are many cases with no time and resources to catch all the errors whether they were unintentional or deliberate. However, it is difficult for judges to avoid using the legal rules in courtesy of results consistently. Avoiding adhering to the rules and regulations makes a judge a target of their colleagues (Gravett, 2017). During the appeal, judges that decide by breaking the rules face a reversal in Supreme Court and are vulnerable to en banc calls.\n\nAlso, the judges are constrained by the political system that checks the judicial excesses, though the factor is often discounted. The political system and process rarely respond to the specific issue in a court ruling, but it does when the contentions affect a significant group. For instance, the Supreme Court experienced intense pressure from Congress on the Civil Right Act of 1991, which they believed was misread (Eliot, 2021; Fine et al., 2017; and Geerling et al., 2017). As such, they decisively and swiftly moved to overrule the decision of the Supreme Court by statutes when they felt that the court erred in its judgment and application of the law. The political pressure constraints the courts in blunter ways; for instance, FDR planned a proposal to clip the jurisdiction of the federal courts on the sensitive matter and pack the Supreme Court (Glöckner, 2016). Also, the voters voted to remove all the three justices in the Supreme Court in California. The voters were persuaded politically that the justices were incompetent and unfair or using their position to engraft political changes in the country. It shows that justices in the Supreme Court are aware that sensitive political matters require deliberate analysis and assessment since it implies their position. The politicians can incite the public against the court's lading to their removal; hence, become sensitive in their reasoning (Harris & Sen, 2018). There is a premise though unspoken that there are less or more objective principles that the law operates, rules that determine the reasoning as established in a court ruling. The judges may not follow these principles; however, they understand their impact on their judgments.\n\nPrinciples like language are integral for judges to use when making their rulings. The choice of words in a statement can have diverse interpretations; thus, judges are inclined to use the appropriate words that defend their reasoning. There are words with immutable and single meanings while others are debatable (Heumann et al., 2019; Hou & Wang, 2020). While reading the statutes, contracts, and regulations in the constitution brings constraints to a judge when deciding on cases. The language used presents an outer boundary that judges use to interpret and apply in their reasoning though there are precise lines with varied meanings. Though fairly strict, the language used is generous to judges though constraints are evident, for example, Article II, Section I, and Clause 5 in the constitution state that persons eligible for the office of the President in the US shall only be a person born and be US citizen (Zygmunt, 2020). The wording in the constitution gives no room or little space for interpretation. The language used offers meaningful and firm constraints on who is eligible for office as a US president though some can debate that an individual born in the US by one parent from a foreign country is ineligible to contest for the office of the predicant in the US. The case in point is that of Obama who faced intense opposition from individuals who believe that he was a foreigner as one of his parents was a Kenyan. At the same time, others can argue that a person born to both American citizens in a foreign nation be considered a citizen by naturalization. Also, another case in point is the Fourth Amendment which proscribes arbitrary and unreasonable seizures and searches (Jiménez, 2021). However, the term ‘reasonable’ raises concern as it is debatable; however, the rulings of courts aren't made in a vacuum. The interpretations of the term ‘reasonable’ reasonable are shared on the notions of personal autonomy and individual privacy. In essence, regardless of the judge's reasoning, warrantless searches on a specific block are not considered reasonable (Jiménez, 2021). The marginal cases present challenges in establishing line-drawing problems, which affirms that language poses constraints on the manner in which courts interpret cases.\n\nMoreover, the judgement presents constraints on how the courts make their rulings. The judges are forced to consider precedence rather than ignore them when making their judgments. They should avoid distinguishing it based on a trivial and unsubstantiated basis. It is difficult to find judges writing their ruling without citing precedents, which offers a constraint on how judges decide (Winter, 2020). Precedence in courts, just like language, provides an opportunity for judges to make their determination; however, dishonesty and judgments are clear when using or burying precedence. Lawyers who fail to provide citations of their arguments with precedence often incense the judges. The lawyers are expected to carefully cite their precedence as an authority for their arguments and properly state the holding cases in a principled manner (Leibovitch, 2016). Lawyers find themselves in a problem when they find two lines of authority are cited within the same subject that provides different lines of discussion. As such, it becomes challenging for lawyers to advise their clients on the rule of law and how to conduct their affairs depending on the judges listening to their case.\n\nPichlak (2020) says that every individual has interests, biases, instincts, and learning that constrain their judgments. Though integral issues, these factors are often ignored in the legal profession. Like all humans, the judges do rely on their instincts as facts and evidence can be masked which affects one’s ability to perceive issues with objectivity (Leiter, 2020). The approach can make a difference when the judges consider their gut feeling or hunches in a case. The facts can be misleading which can lead to the wrong decision by judges; however, when they consider their hunch, they can unmask lies concealed as truth. Nonetheless, it is critical that one is skeptical of their instincts and doubts their leanings as it can result in a wrong conclusion. Establishing the difference between what a person thinks and the decision that they make it difficult to discern (Mańko, 2021). Consequently, it is challenging for interested parties to predict the outcome of a case when they understand the leanings of a judge. However, as realists contend, it is easy to predict an outcome of a case when a person has information about the judge as they are likely to take sides on what they believe. It provides an opportunity for lawyers to fight these impulses rather than yield to them.\n\nJudging is a self-indulgent job with serious ramifications and hazards. The judges are expected to constantly avoid their bias, leaning, persuasion, and instincts when deciding on cases. The judges hold a special role in society as arbiters of cases and custodians of the constitution; hence, their egos and leanings should be abandoned in courts. When trusted, the judges can make good rulings that are acceptable to the public (Weinshall, Sommer & Ritov, 2017). They are called to make judgments on cases and issues that are unpopular to the public; hence, expected to be impartial and reasonable in their rulings. For instance, they are expected to strike out legislation on a matter of public interest, release convicted criminals or terrorists, or allow protests by people perceived as an offending society. The courts expect people to follow their rulings even when they are against their expectations (Menga, 2018). The basis for their argument is that the constitution was created to cause unpopular sacrifices that protect everyone in the society, even when they are a minority.\n\nWestern practices and norms place severe restrictions on the conduct of a judicial officer, which is expected to be fair, rational, and acceptable. Their actions and decisions should be premised on the main tenet, which is the rule of law that should be applied to the same situations and individuals similarly. The rule of law distinguishes judicial decisions since it is premised on law rather than on power. As an arbiter, a judge is expected to be a disinterested arbiter since it promotes corrective justice (Mertz, 2016). The legal profession has internalized the requirement of disinterest as judicial officers should behave in an appearance of approximate pure neutrality. Even when judges are confronted with emotional elements that are challenging and messy, they are sworn to make decisions and determine issues based on customs and laws of the land rather than their private judgment. They are restricted to basing their arguments on popular opinion, pressure from other arms of government, and personal convictions (Mitchell, 2019). Considering the above discussion, judicial conduct premised on personal preferences, popular whims, and political pressure is considered illegitimate.\n\nIn legal education, the practitioners are taught the value of the importance of observing rule of law. The focus on the current legal rules is emphasized to create precedence with policy consideration ignored unless forced through the presence of precedent (Mindus, 2021; Mocan, 2020; and Owens, 2016). Public policy is considered a cost-effective argument as opposed to policy goals such as justice and equality. Unsurprisingly, the judges are hesitant to consider or admit that anything else apart from pure legal stance influences their decisions and rulings. In many instances, the judges are often attacked for judicial activism, which means that they are influenced by ulterior motives other than the law. The judges often note that they are often forced to take counter-majoritarian positions in support of their rulings (Pasquale & Cashwell, 2018). As well, there exists cynicism on whether the legal decisions reflect the reality as the degree of human perfection, just like all customs are probabilities with lived experience. The judges are surrounded by friends, family, life, expectations, and connections that often shape their actions and behaviors when they are off the bench (Pichlak, 2020). A judicial officer with particular outlooks, connections, and positions in society tends to develop stances inclined toward political and social influence. It is difficult to ascertain whether the judges are immune to influence from their religion, political stance, and economic realities when they make decisions and rulings.\n\nIn the long term, an overly rigid judge who fashions adherence to rule of law would be counterproductive to their rightfulness in the courts. The interpretation of Aristotle's idea of justice presents a unique perspective since it emphasized lack of empathy. The decisions of individuals in different contexts affirm that people don't rely strictly on rationality in making choices and judgments as they are often influenced by irrelevant facts such as the presence of random anchors or changes in presentations (Priel, 2020). In the judicial realm, the legal cases should be decided based on relevant facts and law only with minimal extraneous factors. In principle, the decisions of a judge should be devoid of influences such as whether a judge is hungry, exhausted, or the manner in which a case is presented. Still, studies have shown that the judges present biases and fallacies to individuals when they decide on a matter in courts (Quintanilla, Alle & Hirt, 2017). In essence, the decisions of judges involve intuitive and constructive elements rather than those premised on pure rational calculations, which makes their ruling malleable to irrelevant facts.\n\nThe judges play a critical role in protecting and progressing constitution systems and since they are appointed by political actors rather than elected, they experience a democratic deficit. It means that they lack the mandate and authority to make decisions based on their personal policy preferences when making judgments on disputes (Richards, 2016). Though people expect that judges should serve as impartial arbiters, and make a decision based on law and constitution rather than personal preferences, they are constrained by other issues. The basic assumption of the conventional legal reasoning is that judicial discretion is limited, which means that the decisions of judges are constrained by the precedence, text, and reasoned inquiry on the selected drafting officials and the intent of the constitution. Studies that explored the impartiality of judicial decisions concluded that the behavior of judges often decides in favor of judgments that are inconsistent with their policy stance (Ranieri, 2019; Schultz & Kovacs, 2016; Singh, 2018). The focus is on inter-agreement and dissident, which shows that the decisions of judges in some instances border on extra-legal elements. Critics of Supreme Court judgments contend that the institution is political as the justices make decisions that border on and reflect their personal and political values (Smejkalová, 2020). The court is provided with the powers to act on governance issues, which makes the justices at the Supreme Court targets of the impeachment process by Congress. When judges are perceived as partisan, the critics argue that they are guided by political influence and policy preferences (Song, 2019). Besides, the media coverage of the cases in the Supreme Court fuels the perception that political influence plays a role in the determination of cases. Though the press analysis of a court ruling does little to placate the description of judgments as politically influenced, the narrative satisfies political influence.\n\nLegal literature holds that the conventional perspective of legal judgments is made logically and mechanically from the official legal materials such as court cases and statutes, which has often been challenged by legal realism. Legal realism contends that the legal judgments and doctrines are more malleable, less causal of judicial outcomes, and less determinate as opposed to the conventional view that the constraints propose (Spence, 2016). Legal realism believes that extraneous factors and official legal materials influence court rulings. The extraneous factors such as judgment bias, policy and ideological preference of a judge, and intuition guides the judges when making decisions. Legal realism holds the old trope that “justice is what the judges ate for breakfast” (Tejani, 2016). Analysis of the legal decision-making can be explained using theories such as explained by formalism and realism theory, DLA, democratic and Hutcheson Theories (Tejani, 2016).\n\nJudicial decisions are influenced by extraneous factors as opposed to facts and law. The Danziger, Levav, and Avnaim-Pesso (DLA) theory contends that deciding on several cases simultaneously influences the legal outcomes of a judge. The theory premised its finding on the study of 1,112 legal rulings in Israel conducted by the parole board, which handles most of the parole cases in the country (Teichman & Zamir, 2021). They assessed the order of case presentations where they learned that the board handles three cases daily, which are separated by breakfast and lunch breaks. Comparing the first and the last ruling, the study found that favorable outcome drops by 60%. According to the research, the likelihood of a good result decreases from 60% in a first option to zero in a last one (Wei, 2021). As such, the theory established that the judges are influenced by extraneous factors, which they speculate was attributable to depletion of mental cognition. They argued that when the judges make decisions, they become hungry, exhausted, and mentally drained, which forces them to use an effortless and simple strategy to retain the status quo (Troop, 2018). As a result, the number of rejected cases increases since the judges are influenced by their hunger and exhaustion leading to the “irrational hungry judge effect” (Troop, 2018).\n\nJudges face challenging tasks when making decisions with the explosion of fact and indeterminacy of the law. They are floating in the sea of documents, oral courtroom testimony, affidavits, deposition transcripts, and expert opinion that presents a daunting task when deciding cases without reducing and simplifying factual complexity (Bonica & Sen, 2021; Boyd, 2016). The use of case management is adopted by judges to manage the increasing complexity and crushing caseloads; however, consensus holds that law is indeterminate. Among the legal circles, there is a challenge to determine how the judges manage to make informed choices when faced with an explosion of facts. It is difficult to perceive judges as scientists that make decisions like technicians who make choices based on deductive logic (Bozorgmehr & Naseri, 2020). The traditional view is that judicial decision-making should be made based on the application of law and legal rules while others contend that they should make decisions to realize the specific aim or end economic efficiency. Realism argues that judicial decision-making is circumscribed by political power and reasons rather than the law and legal rules (Burns, Dioso-Villa & Rathus, 2017). To understand judicial decision-making, realists contend that deconstructing the reasoning of judges helps to determine hidden meaning and presuppositions such as gender, race, and class that guides it.\n\nHunch theory by Judge Hutcheson on judicial decision-making offers an alternative explanation of the approach embraced by judges when making decisions. The theory acknowledges the importance of judges when making decisions in society and the legal indeterminacy. Hutcheson contends that it is difficult to reduce law into logic as judges are not technicians to determine cases mechanically (Carvacho, Droppelmann & Mateo, 2022). However, the judge notes in the court that judicial decision-making cannot be reduced to politics and made an issue of technical reasoning. The decisions should be premised on an empirical and pragmatic approach, which means that judges are allowed to feel and intuit on decisions that they make. When making decisions, judges must consider all the materials presented and use their hunch or intuition to guide their determination (Chen, 2016). The imaginations of a judge play a role in lifting their mind beyond the conflicting and constricting facts, as well as precedence that can impede just and fair judgments. Through this method, a judge's intellect can be exposed to the full extent of his/her experience, enabling resolution of cases (Chen, 2019). In essence, the hunch theory contends that judges should focus on factual complexity in the presented materials to establish the legal significance without containing and reducing the power of technical reasons.\n\nHunch's theory accounts for the judicial decision-making that is perceived as similar and familiar to indirect and imprecise approaches to deciding on issues that affect individuals in their daily life. The use of terms such as intuition and hunch mean that there is potential arbitrariness and rudeness in the manner that judges make decisions (Chen, Moskowitz & Shue, 2016). The potential challenge with the assumption is to determine whether the judges decide by guessing. It is difficult to presume that judges made a decision based on hunches alone; hence, the case requires epistemological justifications. Anything would be permitted if the hunch theory is validated arguing that judges make decisions by guessing. The adoption of the hunch theory is characterized as advocating for the trope “law is a matter of what a judge had for breakfast” (Cserne, 2020). According to William James’ pragmatism (Olin, 2020), the judges' decisions based on hunches save the arbitrariness, which offers compelling epistemological justification.\n\nThe hunch theory presents a practical explanation and solution for the indeterminacy of the law and the explosion of facts. As such, judges should apply their hunches until they can realize pragmatic conditions established for the justification. These conditions fail to offer a false sense of judicial constraints and legal certainty in the same way legal formalism provides (Dagan, Kreitner & Kricheli-Katz, 2018). Nonetheless, the requirement that judges experience the idiosyncratic sense of certainty on the rulings that they make and test pragmatically on the impact of their hunches pronouncements offers a disciplining effect on the judges as they depend on subjective hunches. Though legal realism is founded on pragmatism, hunch theory contradicts the main tenets of legal realism (Diamond, 2019). The legal realists contend that legal interpretation is premised on law rather than science, which characterizes them as rule skeptics. Legal realism has adopted a scientific approach and narrowed the perception of experience in attempts to reform the law by making it more scientific and rational.\n\nThe democratic theory contends that the framers of the US constitution designed measures that insulated the judicial actors from politics, which helps them to make judgments and pronouncements based on their expert interpretation of the law. Consequently, the judges are given the freeway to make judgments that are consistent with their policy preferences. Segal and Spaeth contend that the voting behaviors in Supreme Court by judges highlight the attitudinal responses triggered by differential case stimuli (Gordon, 2020). The choice of judges is influenced by their policy preferences as established in the litigation. Supreme Court Judges were not democratically responsible for their rulings since they are employed by the institution for rest of their lives (Eliot, 2021). As a result, they make choices that incline them to show their policy preferences. The argumentative nature of the legal system gives room for judges to make decisions premised on the arguments that are compelling to the competing groups. The law permits judges to make decisions without constraining them, which makes them make justifications consistent with their choices after rendering their decisions (Fine et al., 2017). In essence, it shows that they are allowed to take policy preferences rather than base their judgments on legal authority.\n\nThe legal formalism theory contends that there is a pyramid of rules where first principles, middle level, and specific rules guide how judicial decisions are made. It means that when judges are making their ruling, they consider the law and determine the rule that provides the opportunity to make a correct determination (Geerling et al., 2017). The formalism theory contends that for every case, a judge should explore the differences and similarities of previous cases that are classified in taxonomy, which makes the ruling accurate. In law, the basic principles are discerned by induction from rule of law and cases that were premised on principles and decided on from rules (Glöckner, 2016). The critics of legal formalism contend that the life of the law is based on experience rather than logic, which means that the ideologies are not decided by concrete cases. The principles of law are not derived from a watchful analysis of principles and rules, which means that there is a fuzzy overlap where a judge creates a distinction as opposed to discovering it (Gravett, 2017). The differences are often influenced by the sense and perception of a judge, which means that it is often illogical and arbitrary.\n\nThe legal formality theory contends that legal rules and fundamental principles are critical as they offer guidance to judges though insufficient in determining the outcome of cases. The sense of inevitability and certainty as expressed in judicial opinion was considered unjustified by critics (Hamer & Edmond, 2016). However, the legal landscape responded to the failures of formalism as it became dense with intermediate cases. Although two cases can seem different and the distinction is easy to establish, a cluster of cases makes it challenging to trade the difficulty. As such, the determination of cases is based on the preponderance of feeling as opposed to the articulation of reason, rules, and principles (Harris & Sen, 2018). Though the courts are avoiding the idealistic theory that bred formalism, the analytical methods are present. For instance, there is a robust classification of cases as procedural law, property, torts, and contracts versus substantive law. The tenets of formalism theory form the basis of many judicial opinions, which means that the rulings are premised on rigorous analysis of acceptable law and facts (Heumann et al., 2019; Hou & Wang, 2020). The judge discovers the governing principles to help in making the correct decision. There is no room for errors and doubts as the neo-formalist jurisprudence is characterized by self-reported involvement of restraint, singular correctness, and high confidence.\n\nConversely, legal realism theory contends that laws in a country are derived from the existing public policy and social interests, which means that these considerations rather than abstract rules form the basis for the decision of a judge. As aforementioned, the legal realism movement started when law practitioners challenged the existing view that judges are rational arbiters who rely on law and legal rules in making their determinations. When making a decision, judges are expected to determine cases by extension exploring their policy perspectives, principles, and mind before turning to law and legal rules (Jiménez, 2021). Scholars consider legal realism as a controversial theory that, though influential, is often misunderstood. It is a school of thought that distorts and homogenizes court decisions rather than simplifying them. The premise of legal realism is that a judge should develop a preferred outcome for a case before they explore the existing law and legal rules (Leibovitch, 2016). The preferences are based on a non-legal basis such as the public policy preferences, the conception of justice, ideology, the personality of a judge, and the attributes of litigating parties like a poor plaintiff, government, or racial group. After making preferences, the judges look for justification for their choices using the legal rules and law (Leiter, 2020; Mańko, 2021). The legal system is complex and contradictory, which gives judges the freedom to use statutes, cases, canons, maxims, principles, and authorities to back their preferred outcome.\n\nAccording to Mengo (2018), the attribution of the correlation between adjudication and what judges ate for their breakfast by realists and formalists is a longstanding discourse. The judicial decision-making is often linked to frivolous factors such as what they ate with critics contending that these arguments are far-fetched while proponents cite experiments conducted by DLA. The existing literature affirms that there is evidence to an asset that judges make a decision based on extraneous factors as their repeated ruling have an element that favors the status quo, they base their rulings on precedence, which is often contested, and influenced by what judges ate (Menga, 2018). For instance, the proponent of realist theory contends that the judges do make decisions before they back their ruling with the law, facts, and legal rules. It means that personal preferences, policy perspectives, and digestion plays a role in their decision-making process. The parties in the court proceeding influence the decision of a judge as weak, poor, or rich are favored by a judge based on their preference (Mertz, 2016).\n\nThe realists describe adjudication as a process that involves judges responding to the underlying facts of a case when deciding on the case as opposed to the application of reasons and rules. The theory contends that the judges only consider the rules and law after making their decisions. Other realists note that the idiosyncrasies of the personality of judges and extraneous factors influence their decision-making process (Mitchell, 2019; Mindus, 2021). For instance, when judges decide on a case when hungry, they are likely to favor the status quo regardless of the facts and judicial rules. The realists contend that the judicial outcome can be predicted as patterns such as the personality of a judge can help discern how they decide (Mocan, 2020). In essence, the judges make a decision that falls into patterns, which correlate with the factual circumstances in the dispute. The approach that the judges take in response to the factual patterns is governed by the outcome of cases. The realists believe that the courts only enforce the uncodified and prevailing norms that they relate to the underlying factual circumstance. The crux of realists' position is that non-legal reasons explain the decision that judges take (Owens, 2016). The shortcomings of realist findings are that they lack consistency in the use of a scientific approach to criticizing the traditional decision-making process in the courts. Moreover, the theory overemphasizes the submersion of rules and principles, as well as fact-finding. It creates a new form of verbal gymnastics and word enhancement. The critics of realism theory contend that realists perpetuate confrontational and violent society by describing justice as predetermined rather than based on rigorous analysis of facts and law (Pasquale & Cashwell, 2018; Pichlak, 2020; and Priel, 2020). It provides the judges space to make a decision based on their interest, gut, hunch, ideology, or digestion. It encourages judges to act in a manner that perpetuates force, power, and suspicion. The theory contends that judges are faced with immense constraints that make it difficult for them in delivering dependable judgments (Quintanilla, Alle & Hirt, 2017). These constraints include language, precedence, political influence, personal interest, and views of their colleagues.\n\n\nDiscussion\n\nTo determine whether judges are influenced by extraneous factors such as what they take for breakfast, policy preference, their feeling, or cultural contexts, a controlled experiment is necessary. It would be helpful to conduct an experiment to test the variables so that the research question of whether law and judicial rules are the only factors that judges consider before making a ruling (Richards, 2016). The current studies focus on digestion rather than the cognitive biases and heuristics that human beings are subjected to rather than on environmental conditions that influence the decision-making process. The study will bring to an end the debate on whether judges are influenced by extraneous factors apart from the law and legal rules when making their judgments.\n\nThe existing studies show that the realists are perceived as radicals while they term themselves as reformers who wanted to increase the predictability and certainty by explaining the real nature of the decision-making process among the judges (Ranieri, 2019; Schultz & Kovacs, 2016; and Singh, 2018). The realists criticized the perception that judges make a decision based on legal principles and rules, which is considered logical reasoning. They contend that the decisions made by judges have no basis in law and judicial rules alone as non-legal rules and other factors are considered when making judgments. Though they believed in the rule of law, the realists based their arguments on the idiosyncrasy of the law (Smejkalová, 2020). They sought to change and reform the judicial decision-making process by making it certain and efficient. The realism theorists sought the opportunity to reform legal education with the introduction of clinical legal education, which is available in many law schools in the US. The realists are social reformers who sought to make the law a tool for social action; however, they faced stiff objections (Song, 2019). Realists believed that the policy objectives and the legal rules are intimate; thus, social reforms are premised on the knowledge of the elements that drive judicial decision rulings. As such, the realists advocated for an empirical approach to law, which has become a norm.\n\nThe realists believe that the realism theory is a down-to-earth and practical school of thought in touch with reality compared to the inflexible and scientific law of the theoretical model. Most realism theorists were eminent judges and practitioners with experience; thus, they were rarely personified by scientific theories (Spence, 2016; Tejani, 2016). In satirical response to the realism theorists, the continental theorists remarked that what matters is not the prediction of the courts, but what legal rules and law pronounce. Realists argue that judges should base their decisions on explanations and predictions, and not present them as scientific theories. A good theory satisfies two principles including one that explains the large class of observation and makes definite predictions (Teichman & Zamir, 2021). Theories that fail to make predictions should be abandoned in a rational world since failure to satisfy prediction means that it is controlled by a higher power (Teichman & Zamir, 2021).\n\nThe difference between the legal realism and formalism theories is that realists contend that the decision-making process adopted by judges is founded on prediction, which formalists disagree with. If the realists stated that the judging cases are based on adherence to legal rules, they would agree with the formalists (Troop, 2018). The difference between the two theories is the use of prediction, which the realists contend is an integral element in the judicial decision-making process. The realists play an integral role in creating a distinction between decision-making and justification as they established a division between written judgments and judicial opinions and the actual process of making the ruling (Wei, 2021). They contend that judges use the facts and formal judicial rules to justify their choices, which means that formalists avoid the conclusion that judges make decisions based on their personal preference, personality, or hunch. Chief Justice Evan Hughes of the US Supreme Court admitted that judges often make a decision based on emotions with the rational part providing them with the basis for supporting their preference (Weinshall, Sommer & Ritov, 2017). The judges that use convincing language and mechanical judgement are those swayed by dishonesty and emotions but that mask their lawlessness with meticulous justification.\n\nThere is a possibility that the manner in which a judge makes a ruling and their justification coincides, which means that the judicial reasoning and the decision-making often overlap. However, though the ruling may overlap, it does not suggest that it is a perfect pointer to the other. Some scholars believe that the distinction is an inaccurate representation of the judicial decision-making process and decisions (Zygmunt, 2020). The proponents of critical legal studies contend that the actual reasoning of judges reflects their opinions and decision-making styles, which makes judicial opinion couched in a legalistic and formal manner that reflects their thinking. The realists contend that the functional legal system, the concept of law operates outside the court processes, rules, and regulations (Winter, 2020). What the judges do and what the law says in books are different as they use extraneous factors when making their judgments. There are gaps between the enforceable rules and legal valid rules. In essence, how the rules in books are enforced against parties such as racial and ethnic minorities, the political power, and the underprivileged are different, which shows that the cultural environment impacts the decision of a judge (Harris & Sen, 2018). The legal realists emphasize indeterminacy, on which the courts base their arguments and facts that provide judges with interpretative latitude. The judges use the latitude to construe the precedents and statutory provisions. The truth is that the varied interpretations are considered valid and legitimate when they are based on other legal actors rather than a reference to a normative standard (Heumann et al., 2019). As such, the realists depend on the scope of legal indeterminacy when making their arguments against formalism theory. Overall, legal formalists tend to avoid considering that judicial opinion fails to reflect the actual judicial reasoning.\n\nMy appeal is that the independence of the judiciary is protected by trusting in the judges by avoiding frivolous accusations that some findings are based on personal interests, inclination, and the ideology of judges rather than adherence to the rule of law and observing the judicial rules. Encouraging cynicism is dangerous as it causes one to consider their talents in defeating arguments supported by law. Reversing the trend is essential as calling out the actions of those meddling with the appointment and operations of judges will force them to rethink their decisions. Besides commenting on the development of the law, arguing critical cases, helping in the appointment of judges, making government policy, and writing legislation are imperative in reversing the trend where people attack the judges and their reasoning. Commentators should avoid criticizing the self-indulgence of the judiciary as it encourages future generations to disregard the court and constitutionalism. Conversely, judicial officers should ensure that they hold their position morally and legally defensible.\n\n\nConclusion\n\nThe old trope that judges' decisions are based on what they ate as opposed to the law and judicial rules is a contentious discussion. The discussion is centered on whether the judges are influenced by extraneous factors other than the law and judicial rules, which the realists affirm and vehemently denied by the formalists. For formalists, the judicial decision-making process is bound by the law and judicial rules, which are perceived by realists as mechanical and irrational. Though the formalists don't agree with the extreme views of the judicial decision-making process, they nonetheless consider formal rules imperative. On the other hand, the realists contend that the judicial rules are influenced by other factors and that the law and facts are supplementary. There is a consensus among the realists that the rules and law play some role, however, the other extraneous factors are more important. While making a judgment, the judge only considers the laws and rules to support their ruling. Also, for any position, the judges find a legal basis to support their ruling using the formal rules, law, and facts. Though legal realists are perceived as radicals that advocated for unscientific decision-making procedures for judges, they raised attention to the driving force of the judicial decision-making process and factors that influence their rulings.\n\nMy submission is that constitution was created to ensure that everyone feels protected regardless of the persuasion, leaning, instinct, and beliefs of a judge. The principles are designed to help judges make rational and fair judgments; thus, their conduct should generate public trust in the judiciary. I believe that the judges make their determination based on the judicial rules and law as opposed to the political agenda, interests, and attitudes. Lack of trust in the judges can cause points of dissatisfaction when they make unpopular decisions. It brings an impetus for changes that are far-reaching that can impact the lives of people. As such, vilifying judges based on how they voted or made their judgments citing frivolous issues such as their political stand, leaning, ideology, or what they ate can cause harm to the rule of law. For instance, I consider it wrong to throw judges out of office on political grounds rather than their integrity or qualification. The hiring of Chief Justice, Supreme Court judges, and judges in federal courts receive the attention of political players, which is inappropriate since it can affect the lives of people. Installing judges based on their political inclination and ideology can impact the rulings that guide the societal fabric. It offers an opportunity for political players to fiddle with the operations and jurisdiction of the federal courts. When and if the trend continues, it will impact the judicial review that can be limited in scope, affect the removal process, or circumscribe the appointment of judges. Consequently, the judiciary will lose its independence or it will be undermined.",
"appendix": "Data availability\n\nNo data are associated with this article.\n\n\nReferences\n\nAiken J, Shalleck A: Putting the \"Real World\" into Traditional Classroom Teaching. The New Legal Realism: Translating Law-and-Society for Today's Legal Practice. 2016; 1(2): 51–73. Publisher Full Text\n\nAletras N, Tsarapatsanis D, Preoţiuc-Pietro D, et al.: Predicting judicial decisions of the European Court of Human Rights: a natural language processing perspective. PeerJ Comput Sci. 2016; 2(3): e93–e115. Publisher Full Text\n\nAuerhahn K, Henderson JS, McConnell PR, et al.: Are you judged by the residence you keep? Homicide sentencing, attribution and neighborhood context. Criminology, Criminal Justice, Law and Society. 2017; 18(1): 28–51.\n\nBator A: Law and jurisprudence in the face of conflict: Between neutrality and the political. Krytyka Prawa/Critique of Law. 2020; 12(3): 7–31. Publisher Full Text\n\nBonica A, Sen M: Estimating Judicial Ideology. J. Econ. 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}
|
[
{
"id": "158972",
"date": "26 Jan 2023",
"name": "Favio Farinella",
"expertise": [
"Reviewer Expertise International law",
"human rights",
"artificial intelligence and the law."
],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe purpose of this article is to investigate the way in which judicial decisions are constructed. In this regard, the authors present the two major existing theories: legal realism and dogmatic legal interpretation. The first minimizes the normative and prescriptive element of the law and maximizes the empirical and descriptive factors. On the contrary, the dogmatic school is a method of study and legal research and its object of investigation is the norm.\nThe text under analysis seeks to investigate whether judicial decisions are the product of the application of the law on the facts (dogmatic school) or if, on the contrary, other personal and social factors intervene (legal realism school). And in any case, the text aims to demonstrate if such factors exist at the time of the judicial decision, and their real incidence on the judicial decisions.\nThe authors carry out a complete review of the existing doctrine and theories in this regard. Finally, in the conclusion, they offer a well-founded opinion, explaining that although there may be extra-legal factors, the judges mainly make their determination based on the judicial norms and the law without being greatly affected by the political agenda, the interests of the parties to the conflict and other attitudes of third parties.\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": "158971",
"date": "09 Mar 2023",
"name": "Aneesh V. Pillai",
"expertise": [
"Reviewer Expertise Emerging area in International Law",
"Human Rights",
"Cyber Law",
"Consumer Law and Judicial Porcess"
],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe theme of this article is an enquiry about the judicial decision-making process and the factors influencing it. The article proceeds by discussing two well-known theories such as formalism and legal realism. The theory of formalism believes that, the judicial making process always depends upon the facts and circumstances and the application of established legal principles. However, the theory of realism believes that the judicial decision-making process is largely influenced by extra-legal factors. To analyse further, the Article discusses the theories such as Danziger, Levav and Avnaim-Pesso Theory; Hunch Theory; and Democratic Theory. To substantiate the point, the authors have reviewed several kinds of literature on this topic and taken ideas from such literature.\nFrom the analysis, the authors identify that, when the judicial decision-making process is influenced by extraneous factors, there arises a gap between the enforcement of rules and principles. This may adversely affect the protection of interests of racial and ethnic minorities and the underprivileged. The Article identifies that, ‘there is a consensus among the realists that the rules and law play some role; however, the other extraneous factors are more important’. Thus it can be seen that, while making a judgment, the judges give predominant consideration to legal rules and principles; however, other extraneous factors may influence the mind of judges if there arises a situation of personal discretion. Thus the judgements will always have a legal base and is also influenced by judges’ character, social background and other factors. The article concludes by submitting that, fairness in judicial decision-making is an utmost necessity. Hence, the influence of extra-legal factors should be avoided.\nThis article is well written in accordance with the existing standards and legal literatures. It has dealt with the issue of judicial decision making in an elaborate manner. The article cites several relevant references and incorporates the ides from such literatures. Since it has fairly covered all the relevant issues, it can be indexed without any further addition or modification’s.\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/12-9
|
https://f1000research.com/articles/11-156/v1
|
08 Feb 22
|
{
"type": "Research Article",
"title": "Mucosal microbiome is predictive of pediatric Crohn’s disease across geographic regions in North America",
"authors": [
"Rajesh Shah",
"Kristi Hoffman",
"Lee Denson",
"Subramaniam Kugathasan",
"Richard Kellermayer",
"Kristi Hoffman",
"Lee Denson",
"Subramaniam Kugathasan",
"Richard Kellermayer"
],
"abstract": "Background: Patients with Crohn’s disease (CD) have an altered intestinal microbiome, which may facilitate novel diagnostic testing. However, accuracy of microbiome classification models across geographic regions may be limited. Therefore, we sought to examine geographic variation in the microbiome of patients with CD from North America and test the performance of a machine learning classification model across geographic regions. Methods: The RISK cohort included 447 pediatric patients with CD and 221 non-inflammatory bowel disease controls from across North America. Terminal ileum, rectal and fecal samples were obtained prior to treatment for microbiome analysis. We divided study sites into 3 geographic regions to examine regional microbiome differences. We trained and tested the performance of a machine learning classification model across these regions. Results: No differences were seen in the mucosal microbiome of patients with CD across regions or in either the fecal or mucosal microbiomes of controls. Machine learning classification algorithms for patients with CD performed well across regions (area under the receiver operating characteristic curve [AUROC] range of 0.85-0.91) with the best results from terminal ileum. Conclusions: This study demonstrated the feasibility of microbiome based diagnostic testing in pediatric patients with CD within North America, independently from regional influences.",
"keywords": [
"Crohn’s disease",
"microbiome",
"inflammatory bowel disease",
"machine learning"
],
"content": "Introduction\n\nCurrently, our understanding is the intestinal microbiome plays a role in the pathogenesis of inflammatory bowel disease (IBD),1 and specifically Crohn’s disease (CD).2 The RISK consortium found significant differences in the taxonomy of the mucosal and fecal microbiomes of pediatric, treatment naïve patients with CD compared to non-IBD controls.3 Similar results were demonstrated in a longitudinal study of adult patients with IBD with an emphasis on disruption of the microbiome during periods of disease activity.4\n\nBased on an altered microbiome composition in patients with CD, microbiome signatures may be utilized as a diagnostic biomarker. From the original RISK publication,3 the addition of microbiome data to clinical information improved the performance of their classification models for CD. Similarly, Pascal et al. showed microbiome classification models for CD were accurate and performed well across 4 countries in Europe (Spain, Belgium, the UK and Germany).2\n\nRecent data suggested that geographic bias, however, may limit the validity of microbiome based diagnostic models. He et al. studied 7,009 individuals from 1 Chinese province with 14 districts to determine regional differences in the microbiome.5 They found strong associations between microbiome composition and host district, which translated into decreased model performance when classifying metabolic diseases across districts. However, they acknowledged that other diseases, such as CD, could not be studied due to a limited sample size. Therefore, we sought to examine differences in the intestinal microbiome of pediatric patients with CD by region and to determine if geographic bias hinders the performance of a machine learning classification model across regions in North America.\n\n\nMethods\n\nA post hoc analysis of the RISK cohort was performed. The RISK cohort was a multicenter study that enrolled treatment naïve pediatric patients aged 3 to 17 years with CD and non-IBD controls from 28 sites with the United States and Canada from 2008 to 2012.3 All patients had symptoms suggestive of CD, including abdominal pain or diarrhea that prompted evaluation with a colonoscopy with biopsies from the terminal ileum and rectum. A subset of patients also provided fecal samples. Patients were either diagnosed with CD, based on endoscopic appearance and histology, or a non-inflammatory etiology for their symptoms, which served as the non-IBD controls. Full inclusion and exclusion criteria for the RISK cohort have been described in the original publication.3 In total, 447 patients with CD and 221 non-IBD controls were included in the original publication and they provided a total of 1,321 samples, including 630 ileal, 387 rectal and 304 fecal samples.\n\nIRB approval was not required for this study, as deidentified data was used and consent was previously obtained from participants when they enrolled in the RISK cohort study.\n\nAge at diagnosis, sex, race, disease phenotype, and treatment center were examined. To evaluate the influence of geography on microbiome composition, we grouped the treatment centers into 3 subjective regions based on overall geography (North-East, South-East and West, Figure 1A).\n\n16Sv4 rRNA gene analysis was performed in the original cohort study using the Illumina MiSeq platform. For our analysis, the original biom table was obtained and rarefied to 3,441 sequences per sample. This rarefaction depth was chosen to retain the maximum number of samples and preserve the most amount of sequencing data per sample. The alpha and beta diversity and taxonomic composition of the terminal ileum, rectum, and fecal microbiomes were evaluated using the ATIMA interface version 1.0 available through the Baylor College of Medicine Alkek Center for Metagenomics and Microbiome Research. ATIMA is a graphic user interface that allows users to provide a biom table and mapping file for microbiome analysis. To adjust for potential confounding, MaasLin was used to control for variations in age at diagnosis, sex, race, sample type and geographic region.6\n\nFinally, we sought to develop a machine learning model to evaluate the accuracy of a microbiome model to identify patients with CD across different regions. A random forest machine learning model was trained on patients from the North-East and tested in the South-East and West using the R package healthcare.ai version 2.5.0 with the default settings. The healthcare.ai package is an open-source R package that allows for data cleaning, manipulation, imputation, tuning of models and evaluation of model performance. Visualization of model performance with AUROC metrics was done using the R package pROC version 1.18.0.\n\n\nResults\n\nBased on the terminal ileum biopsies retained after rarefaction, we included 227 patients with CD and 165 controls with a mean age of 12.2 and 12.1 years, respectively. Approximately half of patients with CD and controls were male (58.6% and 53%, respectively) and a larger proportion of patients with CD were Caucasian compared to controls (78.9% and 68.7%, respectively). Since microbiome composition can be influenced by the presence of stricturing/fistulizing disease7 and these patients present less of a diagnostic challenge, they were excluded from our analysis to create a consistent population with an inflammatory phenotype. After separating into regions, 182 patients were in the North-East, 33 in the South-East and 12 in the West with CD, and 106 patients in the North-East, 43 in the South-East and 16 in the West without IBD.\n\nFor patients with CD, no significant differences were found in alpha and beta diversity of the ileal and rectal mucosal microbiome by geography. However, PCoA plots of unweighted and weighted beta diversity (Figure 1B) determined through the Bray Curtis metric revealed significant differences in fecal samples. In controls, no significant differences were found in alpha and beta diversity of the ileum, rectum or fecal samples. In the South-East, patients with CD had a relative increase in Fusobacteria and Bacteroidetes with a decrease in Actinobacteria and Firmicutes in fecal samples compared to the other 2 regions. This corresponded to an increase in the genera Bacteroides and Fusobacterium with a decrease in Bifidobacterium and Lactobacillus. However, after adjustment with MaasLin, Erwinia was the only genus associated with geographical variation in patients with CD. Specifically, fecal samples from CD patients in the South-East had increased abundance of Erwinia compared to other geographic regions in North America (q=0.04).\n\nRandom forest models across sample types performed well (Figure 1C). The best performance occurred with ileal samples (North-East AUROC 0.89, South-East AUROC 0.85 and West AUROC 0.91). The rectal (North-East AUROC 0.87, South-East AUROC 0.83, West AUROC 0.76) and fecal (North-East AUROC 0.82, South-East AUROC 0.85, West AUROC 0.74) samples performed well, but experienced decreased performance in the West. Comparing the models, those for ileum and rectum shared OTUs discriminating CD, which included members of the Lachnospiraceae and Clostridiaceae families and the genus Blautia. Intriguingly, ileal biopsies and fecal samples shared top CD-discriminating OTUs from the Erysipelotrichaceae family and Haemophilus genus, which were not present between rectal biopsies and fecal samples.\n\n\nDiscussion\n\nOur results indicate that CD influences mucosal microbiome composition to a greater extent than geography in pediatric patients from North America. Machine learning classification models performed well across the regions, despite minor differences in the fecal microbiome of CD patients. Differences in microbiome composition are known to vary across populations in healthy cohorts8,9 and in patients with metabolic syndrome.5 Yatsunenko et al. showed Westernization may influence fecal microbiome composition by comparing samples from subjects in the US, Venezuela and Malawi.8 Similar patterns were seen by Pasolli et al. when they examined metagenomes from 9,428 samples from 32 countries and noted significant differences in the metagenomes of Western populations.9 Together, these studies demonstrated microbiome composition varies across populations, however, they did not address microbiome differences within countries. To that end, He et al. studied a single province in China and noted differences in microbiome composition between its districts.5 This suggested, as has been previously reviewed, that a vast number of environmental factors may play a role in shaping the microbiome and may limit the accuracy of microbiome classification models.10\n\nOverall, our classification models performed well across regions and is consistent with prior reports. Using 2,045 fecal samples taken from patients with IBD and non-IBD controls across 4 European countries, Pascal et al. showed that a microbial signature could be used to discriminate patients with CD from non-IBD controls with an overall sensitivity of 80% and specificity of 94%.2 In a separate cohort, Franzosa et al. used metagenomics and metabolomics to distinguish IBD patients from non-IBD patients also with high accuracy.11 Our findings in pediatric CD are consistent with these results and demonstrate the feasibility of using microbiome classification models to accurately diagnose CD without geographic bias within North America.\n\nDespite the limitations of our study, our classification models performed well. We were unable to adjust for additional confounders of microbiome composition, such as diet and supplement intake.12 However, even without this information, our models based on ileal biopsies performed well. Additionally, we noted a decrease in model performance for fecal samples and in the West, but this may be linked to a smaller sample size, which is known to hinder the performance of machine learning models. Further work with larger cohorts and different control groups will be needed to fully determine whether microbiome machine learning models can support the diagnosis of CD in children without geographical bias, and if non-invasive testing with fecal samples is feasible.\n\nIn summary, machine learning models can distinguish patients with CD from non-IBD controls without geographic bias in North America. Further development of microbiome machine learning models to diagnose CD may be warranted.\n\n\nData availability\n\nNCBI BioProject: human gut metagenome. Accession number PRJNA237362; https://identifiers.org/NCBI/bioproject:PRJNA237362.\n\nThe underlying clinical data used for this study is available through the RISK consortium. Consortium approval was required to access de-identified patient data and requests can be placed through the Crohn’s and Colitis Foundation IBD Plexus Initiative (www.crohnscolitisfoundation.org/research/granst-fellowships/ibd-plexus).",
"appendix": "References\n\nHuttenhower C, Kostic AD, Xavier RJ: Inflammatory Bowel Disease as a Model for Translating the Microbiome. Immunity. 2014; 40(6): 843–854. PubMed Abstract | Publisher Full Text\n\nPascal V, Pozuelo M, Borruel N, et al.: A microbial signature for Crohn’s disease. Gut. 2017; 66(5): 813–822. PubMed Abstract | Publisher Full Text\n\nGevers D, Kugathasan S, Denson LA, et al.: The Treatment-Naive Microbiome in New-Onset Crohn’s Disease. Cell Host Microbe. 2014; 15(3): 382–392. PubMed Abstract | Publisher Full Text\n\nLloyd-Price J, Arze C, Ananthakrishnan AN, et al.: Multi-omics of the gut microbial ecosystem in inflammatory bowel diseases. Nature. 2019; 569(7758): 655–662. PubMed Abstract | Publisher Full Text\n\nHe Y, Wu W, Zheng H-M, et al.: Regional variation limits applications of healthy gut microbiome reference ranges and disease models. Nat. Med. 2018; 24(10): 1532–1535. PubMed Abstract | Publisher Full Text\n\nMorgan XC, Tickle TL, Sokol H, et al.: Dysfunction of the intestinal microbiome in inflammatory bowel disease and treatment. Genome Biol. 2012; 13(9): R79. PubMed Abstract | Publisher Full Text\n\nKugathasan S, Denson LA, Walters TD, et al.: Prediction of complicated disease course for children newly diagnosed with Crohn’s disease: a multicentre inception cohort study. Lancet. 2017; 389: 1710–1718. PubMed Abstract | Publisher Full Text\n\nYatsunenko T, Rey FE, Manary MJ, et al.: Human gut microbiome viewed across age and geography. Nature. 2012; 486(7402): 222–227. PubMed Abstract | Publisher Full Text\n\nPasolli E, Asnicar F, Manara S, et al.: Extensive Unexplored Human Microbiome Diversity Revealed by Over 150,000 Genomes from Metagenomes Spanning Age, Geography, and Lifestyle. Cell. 2019; 176: 649–662.e20. PubMed Abstract | Publisher Full Text\n\nGupta VK, Paul S, Dutta C: Geography, Ethnicity or Subsistence-Specific Variations in Human Microbiome Composition and Diversity. Front. Microbiol. 2017; 8: 1162. PubMed Abstract | Publisher Full Text\n\nFranzosa EA, Sirota-Madi A, Avila-Pacheco J, et al.: Gut microbiome structure and metabolic activity in inflammatory bowel disease. Nat. Microbiol. 2019; 4(2): 293–305. PubMed Abstract | Publisher Full Text\n\nLewis JD, Chen EZ, Baldassano RN, et al.: Inflammation, Antibiotics, and Diet as Environmental Stressors of the Gut Microbiome in Pediatric Crohn’s Disease. Cell Host Microbe. 2015; 18(4): 489–500. PubMed Abstract | Publisher Full Text"
}
|
[
{
"id": "150774",
"date": "05 Oct 2022",
"name": "Jonathan Braun",
"expertise": [
"Reviewer Expertise Immunology and microbiome in IBD"
],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nRegional contribution to microbiome composition is an important and less-assessed factor in refining conclusions about disease association and microbiome. This study is useful in addressing this question in the context of US regions and the landmark RISK cohort study of Crohn's disease.\nMy issues with the paper are minor. It references several assessments with negative associations, for which data is not shown. It is fine to do so, but the methods should be briefly noted: what was the alpha diversity metric(s); and, is the unweighted beta diversity (noted in context of Figure 1, which is weighted) a data-not-shown finding, or a clean-up needed in the text?\nFigure 1B has \"lines\" connecting dots in the PCoA plot. What do these lines represent?\n\nIs the work 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": "8886",
"date": "04 Jan 2023",
"name": "Rajesh Shah",
"role": "Author Response",
"response": "We greatly appreciate the comments and favorable review. To address the raised points, we limited the presentation of negative findings to allow us to focus on positive findings. In terms of the metrics used, we used the Shannon metric to measure alpha diversity and the Bray-Curtis metric for beta diversity. The lines presented in Figure 1B refer to the centroid of each Region."
}
]
},
{
"id": "150775",
"date": "14 Oct 2022",
"name": "Ranko Gacesa",
"expertise": [
"Reviewer Expertise Bioinformatics",
"Microbiome",
"Artificial intelligence",
"Inflammatory Bowel 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\nAuthors examined consistency of microbiome-trained machine learning models for prediction of Crohn's disease across US geography. They demonstrated that models trained on one of geographic regions perform well on ileal samples, but perform less consistently for fecal and rectal samples.\nStudy is relevant (as the loss of predictive power of microbiome models due to geographic variation was demonstrated before in the Chinese population) and methodologically sound. The study would, however, benefit from sharing of used codes, produced models and other technical details to facilitate the reproducibility and comparison with other, related, 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?\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": "9002",
"date": "04 Jan 2023",
"name": "Rajesh Shah",
"role": "Author Response",
"response": "We are happy to provide the underlying code that was used to generate the data for this study in a supplement to facilitate reproducibility."
}
]
}
] | 1
|
https://f1000research.com/articles/11-156
|
https://f1000research.com/articles/11-880/v1
|
02 Aug 22
|
{
"type": "Research Article",
"title": "A model of coercive control in higher education: a qualitative study",
"authors": [
"Maria Jakovljevic",
"Nkopodi Nkopodi",
"Nkopodi Nkopodi"
],
"abstract": "Background: A growing body of research indicates that psychological coercive control poses a threat in academic environments. Little is known, however, about the process, the dynamics, and the phases used to impose silently a variety of non-violent assaults on students and academics. A lack of awareness of coercive intimidation and psychological coercive control obstructs a student’s path to academic achievement, which can have an impact on his or her emotional and mental well-being and diminishes the prosperity of society.\n\nMethods: A methodological selection and review of the scientific literature, theories, and practice on psychological intimidation, coercive control, and systems thinking has been employed in this study. A comprehensive reflective analysis and critical synthesis of the relevant scientific literature were conducted to gain insight into the design of a model of psychological coercive control applicable to educational environments.\n\nResults: This article identifies gaps in research theory and practice and examines critical issues of intimidation and psychological coercive control that is relevant to educational contexts. The article proposes a conceptual model of psychological coercive control as a direction for further research.\n\nConclusions: Adequate awareness, models, and training programmes in relation to coercive infiltration are missing at higher education institutions. There is an urgent need for a curriculum change that may serve to promote support systems thinking and security awareness in educational environments.",
"keywords": [
"Higher education",
"coercive control",
"intimidation",
"bullying",
"systems thinking",
"coercive behaviour"
],
"content": "Introduction\n\nThere is a lack of research that explores the many forms of psychologically destructive behaviour that damage or depersonalise others (Galtung, 1990; Anderson & Bushman, 2002; Cialdini, 2006; Sentse, Kiuru, Veenstra, & Salmivalli, 2014). Psychologically malicious behaviour includes any action, verbal or non-verbal, oral or written, physical or non-physical, active or passive, public or private, individual or institutional/societal, human or divine, in whatever degree of intensity that abuses, violates, injures, or kills (Dahlberg & Etienne, 2002; Buss, 2005).\n\nIntimidation has been recorded as one of the most common types of psychologically destructive behaviour currently present at university campuses (Olweus, 1993; Low, Polanin, & Espelage, 2013). Intimidation refers to a threatened sensation and feeling discouraged or afraid (Karim & Duchcherer, 2014). Intimidation is a deceptive type of social stimulus aimed at influencing the behaviour, emotions, and perceptions of targets (Farmer, Nizeye, Stula & Keshavjee, 2006; Mahon, 2007). General lack of awareness of psychologically harmful behaviour creates a vulnerability that lends itself to behaviours of intimidation targeted against university personnel (UN, 1989; Olweus, 1993). This may be caused by a lack of a systematic theory regarding the structures and processes involved in coercive control (Van Dijk, 2006, p. 359; Sweeney & Sterman, 2000; Cabrera, Colosi, & Lobdell, 2008; Plate, 2010; Ross & Jon, 2015).\n\nResearch findings indicate that the processes and dynamics of coercive intimidation in higher education contexts have not been systematically examined. Van Dijk (2006, p. 361) explains that during intimidation “the act of persuasion doesn’t completely block the interlocutors of their free belief of action, whereas during coercive control a process of a force is applied in a hidden way and the recipients are unable to understand the real intentions or to see the full consequences of the beliefs or actions advocated by the manipulator”. Coercive control involves using social influence to change behaviours, beliefs, and emotions, having knowledge of the victims’ vulnerabilities, and disguising destructive intentions through a pleasant appearance (Simon, 1996; Mitnick & Simon, 2003; Braiker, 2004; Packard, 2007).\n\nBased on the above discussion, the main purpose of the study is to explore underlying issues of different forms of intimidation and coercive control in higher education, so as to suggest a model for empowering students and academics in terms of awareness, understanding, prevention, and rectification of consequent damages. This has led to specific research objectives, namely: (1) to examine current theory and practice on intimidation and coercive control; (2) to critically analyse the association between systems thinking and psychological coercive control and intimidation; and (3) to create a model of psychological coercive control that will inspire academics to take collaborative steps to respond to intimidation and manipulative influence at their institutions. The emerging questions set in this paper are:\n\nResearch Question 1: What are the major components of a model of coercive control (MCC) in higher educational contexts?\n\nResearch Question 2: What are the stages of coercive control at institutions of higher education?\n\nThe remainder of this article is structured as follows: The “Research methodology” section covers the qualitative research design employed in this study. The “Theoretical framework for a model of psychological coercive influence in higher education” highlights multiple forms and challenges of intimidation, psychological manipulation, systems thinking and current preventive measures. “A model of coercive control” (MCC) section presents the components, stages, dynamics, and the procedure and flow within the model. The “Discussion” section outlines the discussion with the answers to research questions. The paper ends with “Conclusions” and “References”.\n\n\nMethods\n\nThis is a reflective study that is based on several papers and other scientific material on coercive intimidation in higher education and that offers a critical appraisal to answer formulated research questions. Reflection specifies self-reflection, critique, and the impact of researchers’ thoughts in producing research outcomes in relation to hidden forms of coercive control in educational contexts. The critical reflection that is adopted in this study is about interpreting one’s own assumptions and about critically evaluating one’s own perspectives from those of others (Alvesson & Sköldberg, 2000), and it is also about eliciting informed options about the ideas of others (Fook, 1996, 2002, 2004; Koch & Harrington, 1998). The study was conducted from October 2019 in the South African higher education environment.\n\nThe researchers of this study offer their own thoughts and reactions on the literature and the existing body of knowledge that may contribute towards a critical analysis of current constructs on coercive control in educational environments (Rosaldo, 1994; Smyth & Shocklock, 1998; Rossiter, 2005; Morley, 2008). Educators generally agree with the literature that suggests prevalent bullying techniques and preventive measures (Scott, 2014). It is known that people are becoming more easily and willingly pacified by subconscious manipulation techniques (Packard, 2007). The researchers have, however, considered whether there might be other hidden methods of manipulation and alternative strategies for challenging the educational system to improve an awareness of this widely spread phenomenon.\n\nOne of the ways to challenge and to change psychological manipulation attempts is through critical reflection of contemporary thoughts in literature (Alvesson & Sköldberg, 2000). Furthermore, the present research intends to contribute towards an agenda of coercive influence in educational environments, and both critical and constructivist research paradigms were useful to support this goal. A critical and constructivist approach to research is less determined by methodology and places a superior emphasis on the “philosophical and epistemological underpinning of the research” (Denzin & Lincoln, 2000, as cited in Morley, 2008).\n\nA critical reflection may free us from “fixed and potentially restrictive ways of thinking and may indicate avenues for change” (Fook, 1996, p. 199, as cited in Morley, 2008). If we change the ways, we construct our decisions in relation to the problem of hidden coercive influences in educational contexts and we may generate a new model that has not previously been considered. Based on the insight of the critical reflection of current research opinions, we lay down a model of coercive manipulation. Our critical insight accounts for ways of early detection of coercive manipulation, and the model presented will help to highlight ethical challenges and to encourage multidisciplinary research of the systems theory.\n\nTraditional literature reviews, according to Snyder (2019), have been supplemented by critical reflection rather than by following a systemic methodology. The search strategies were initially drafted by the authors and discussed with an experienced librarian, and then they were further refined through frequent digital team communications (Jakovljevic, 2022). The search was conducted using electronic methods and by reviewing the catalogues of published papers. In addition to the Unisa library, the authors searched Google Scholar, EBSCOhost, WoS, PubMed, the National Library of Medicine, and Academia.edu, as well as the official websites related to the relevant topics. By means of the “snowball” technique, other papers were identified by examining physically the bibliographies of published papers (Snyder, 2019).\n\nThe search was an iterative process as the authors became more familiar with the databases, and the searches were modified in response to the findings that evolved as additional search phrases emerged. Eligibility search criteria focused on the core concept, the phenomena of psychological manipulation and the issues of coercive control (e.g., coercive control in higher education, intimidation, bullying, systems thinking, coercive behaviour, manipulative channels, and discourse techniques) in line with the research questions. The following search phrases, ‘coercive control’, ‘intimidation’, ‘bullying’, ‘systems thinking’, and ‘coercive behaviour in higher education’, were chosen.\n\nAccordingly, studies were included if they were assessing different aspects of intimidation and coercive control. With reference to coercive control in higher educational contexts, there were, however, few scientific papers. In total, 60 papers were included in this study. The subsequent criteria were used to exclude written scientific material, in line with the need for sampling saturation: these were the scientific material that did not include explanations of the phenomena of coercive manipulation and the scientific material that did not focus on the issues of psychological coercive control, e.g., economic aspects, primary educational contexts, and management and policy aspects.\n\nThe authors discovered that any further search, which was determined by eligibility criteria, could not necessarily add anything to the phenomena of psychological manipulation or to the theoretical framework for the model creation and that it could be “counter-productive” (Strauss & Corbin,1998; Ritchie, Lewis & Elam, 2003). Since our sampling strategy provided relevant textual scientific sources of evidence, in accordance with the aim of the study and research questions, the sampling saturation was reached.\n\nThe literature was critically analysed and reflectively synthetised into several major sub-topics (Jakovljevic, 2022) that were evaluated as the most relevant in explaining the phenomenon of psychological coercion in higher education environments (Bertalanffy, 1967; Kothari, 2004). The synthesis and analysis of the theoretical concepts and a combination of practical and reflective experiences enabled the authors to produce a model of psychological coercive influence applicable to higher education.\n\nQualitative data analysis was performed through the following stages: preparation, organization, review of data, creating initial descriptive codes, reviewing descriptive codes, combining into themes and the presentation of themes in a cohesive manner (Alvesson & Sköldberg, 2000; Ritchie, Lewis & Elam, 2003).\n\nIt was necessary to become familiar with data by reading though the initial transcripts and thinking about the narrative that was voiced within the data (Ritchie, et al., 2003). The first stage of data analysis involved the process of initial coding, whereby each line of the relevant textual data was considered to identify an initial category (Noble & Smith, 2013). Coding, or the process of organizing and sorting qualitative data, was the second step in the data analysis. Codes were used to retrieve and categorize data that were similar in meaning, so that the researcher could quickly detect emerging themes.\n\nIn order to organize, structure, and interpret the data into meaningful themes, descriptive colour coding, which allowed the research to be reflexive, critical, and rigorous in terms of the written source of the data, was used in the study (Jakovljevic, 2022). The authors coded the data, according to different colours of highlighted markers, each representing a different category, and they kept in mind the research questions; this helped, in the later stages, to develop themes in the data (Strauss & Corbin, 1998).\n\nThe coding process involved searching the text for similar ideas and concepts and then marking those elements with code colours (Stuckey, 2015). This coding method made it easier to identify any patterns by comparisons of similar concepts that were used in the derivation of themes. Once coding was completed, the collected data were examined to formulate themes and draw conclusions in line with research questions (Noble & Smith, 2013; Bowen, 2009). For example, with “bullying”, it was necessary to decide which specific words or phrases that were coded in this category were related to bullying (e.g., intimidation, harassment).\n\nDocumentary analysis of textual data consisted of examining, categorizing, and tabulating data to address the aim of the study (Tesch, 1990; Bowen, 2009). A constant comparative method was applied on data within the source of evidence and between sources of evidence. The selected written data sources were critically analysed and reflectively synthetized into nine final themes: Bullying and harassment are prevalent forms of intimidation; Antibullying programs are widely applied for concurring bulling and intimidation; A lack of standardization and common regulations on antibullying measures; Communication act of dominance, positioning and language discourses encourage psychological manipulation; A systematic theory, a framework, the structure and processes of manipulation are absent in higher education; Predominant subconscious and deceptive nature of psychological manipulation; Education of maturity, courage, resolution and systematic reflection as preventive measures; Enabling a comprehension of coercive control through teaching systems thinking; they were evaluated as the most relevant in explaining the phenomena of psychological manipulation in higher education environments and as a basis for the model design (Smyth & Shocklock, 1998; Alvesson & Sköldberg, 2000; Ritchie, et al., 2003).\n\nThe techniques to enhance trustworthiness were peer/colleague examinations, the statement of the researcher’s biases, and the commitment of the researcher to the study. The strategies for internal validity, such as making inferences and analytical pattern matching, were followed in this study.\n\nA rich description of the researched phenomenon, which was embedded in system thinking as a theoretical perspective, contributed to the external validity of this study. The process of data collection and analysis was done simultaneously and in an iterative way. Triangulation of data sources enhanced trustworthiness of this qualitative study because multiple sources were gathered, and data were compared through in-depth thematic analysis in an iterative way.\n\n\nTheoretical framework for a modsel of psychological coercive influence in higher education\n\nIntimidation is a form of non-violent behaviour that utilises prejudice and discrimination, based on race, colour, national origin, ancestry, gender, religious practice, age, disability, or sexual orientation, against others; it is often reflected as angry expressions, emotional and verbal abuse, and embarrassment (Anderson & Bushman, 2002; Veenstra, Dijkstra, Steglich, & Van Zalk, 2013; Mononen, 2017). Intimidation is an aggressive behaviour against weaker victims over time, performed in a repeated manner by an individual or a group (Olweus, 1978; Anderson & Bushman, 2002). Incidents of intimidation aim to destabilise educational institutions (Van Dijk, 2006; Karim & Duchcherer, 2014).\n\nHumans, with their psychological, biological, and social features and their fear of ridicule, isolation, and exclusion (Dahlberg & Etienne, 2002; Paulhus & Williams, 2002), naturally subdue themselves to intimidation practice. This is advocated by the fact that humans’ intellectual capacity deteriorates if there is no adequate social stimulation (Paulhus et al., 2002). Accepting a submissive stance progress, however, into intimidation that is not acceptable (Foucault, 1977; Mitnick & Simon, 2003). Moreover, because of cultural and linguistic differences between people of Eastern and Western countries, intimidation practices differ (Menesini & Salmivalli, 2017).\n\nIntimidation, in the form of bullying on campuses, is the main cause of non-violent maltreatment that results in the weakening of capacity, motivation, and self-confidence, and even in depression among students (Scott, 2014; Meriläinen, Sinkkonen, Puhakka, & Kaäyhko, 2016). Most curricula are, however, hampered by current pedagogical methods of teaching, learning, information sharing, and exchange, with little exploration of hidden intimidation issues among students and academics (Adorno, 1998). What are the predominant forms of intimidation practice?\n\nBullying is a systematic abuse of power by peers, usually towards weaker individuals or smaller groups, that causes short- and long-term detrimental outcomes, such as negative emotional arousals and damage to physical, psychological, or economic well-being (Musselman, McRae, Reznick, & Lingard, 2005; Karim & Duchcherer, 2014; Van Noorden et al., 2016; Menesini & Salmivalli, 2017). Bullying is the practice of forcing another party to act in an involuntary manner, and it uses threats or force, which violates the freewill of an individual and, in a way that is contrary to their own interests, it induces a desired response (Olweus, 1978, p. 199; Scott, 2014). This behaviour is predominantly directed towards students with disabilities, those suffering from obesity, sexual minorities, and those belonging to different ethnic or religious groups (Ojo, 1999; Vitoroulis & Vaillancourt, 2015; Burrowes, 2016).\n\nUsually, modelling plays a role in bullying, as students are more likely to increase bullying behaviour when they model themselves on peers who are galvanised by bullying (Van Noorden et al., 2016). During the school years, bullying and harassment are the most common expressions of non-violent behaviour (Karim & Duchcherer, 2014; Van Noorden et al., 2016). Harassment as intimidation behaviour may comprise, but is not limited to, epithets, derogatory comments, blocking movement, or any physical or verbal interference with movement and visual insults (Musselman et al., 2005; CAIR, 1996).\n\nDilmac (2009) indicates that 22.5 per cent of students engage in cyberbullying behaviour, and the researchers, Al-Raqqad, Al-Bourin, Al Talahin, and Aranki in 2017, indicated that bullying and harassment have caused lower academic achievements in the private and government education sector in Jordan. According to the Progressive Teachers Union of Zimbabwe (PTUZ) report (2002), there has been widespread intimidation of teachers and students in Zimbabwe. The Canadian Association of Interns and Residents (CAIR, 1996) has been concerned about intimidation and harassment issues in post-graduate medical education for several years. From fragmental analysis of harassment, bullying, and other forms of intimidation, one cannot understand the hidden agendas that highlight coercive influence in educational environments (Packard, 2007; Veenstra et al., 2013; Karim & Duchcherer, 2014).\n\nThe rise of many forms of intimidation seems to have obscured other manifestations of psychological non-violent behaviour, such as manipulation, that are coercively echoed in educational contexts because of economic, social, cultural, and psychological factors (Buss, 2005; Sentse et al., 2007). One of the most pervasive and most dangerous forms of non-violent behaviour are those that are often hidden from view in a coercive manner (Packard, 2007; Low et al., 2013).\n\nAdvancements in Information Communication Technology (ICT) have rapidly galvanised coercive control as a deceptive type of social stimulus that aims to influence the behaviour, emotions, and perception of youths, triggering frustrations, reduced enthusiasm, and psychological paralysis to work (Thaler & Sunstein, 2008; Cialdini, 2006; Beale & Hall, 2007; Van Dijk, 2016).\n\nPsychological coercive control is a method of skilful deception, with cunning, prejudicial, or discreet manoeuvres and setups in a clandestine manner, with the purpose to control or dominate individuals or groups (Mahon, 2007; Stanciugelu, 2010; Cialdini, 2006). Perpetrators apply the law of attraction and appeal, in terms of his or her superior skills, capabilities, and accomplishments that attract a weaker individual or groups (Simon, 1996; Braiker, 2004; Tepper et al., 2008; Stanciugelu, 2010).\n\nAlthusser (1970) points out that implying engineered, coercive, or authoritative social control and surveillance can impair emotional, intellectual, and economic development of the individual, institutions, and society. The manipulator creates a relation of trust or suspicion, but “the recipients lack the specific knowledge that might be used to resist manipulation”, and they have an inability to understand the actual purpose, or to realise the full consequences, of the act (Wodak, 1987, as cited in Van Dijk, 2006, p. 360; Rojo & Van Dijk, 1997; Packard, 2007). The hidden persuaders or coercive intimidators propose to the individual a certain state of mind, which is personal, with the intention to influence the behaviour of the individual against his or her will and interests, on a subconscious level (Van Dijk, 1996; Cialdini, 2006; Packard, 2007; Stanciugelu, 2010).\n\nA manipulation channel can be media, technology, or even a whispering campaign (Stanciugelu, 2010). Discourse interactions, as forms of informing, teaching, and persuasion through coercive manipulation, influence control of cognition and actions (Van Dijk, 2006, p. 366). Consequently, this subtle communication discourse involves a need for a “positioning” as a general human behaviour, since people need to see themselves in terms of dominance, to activate representations, emotions, and social definitions of superiority, competence, and success (Mucchielli, 2003; Tepper et al., 2008; Stanciugelu, 2010).\n\nThere are multiple discourse techniques (narratives) used to manipulate, including projection of guilt, imposing uncertainty, creating unresolved tension, putting on a defensive stance, playing the role of authority without responsibilities or vice versa, declarations of enslavement, false remorse, fear mongering, gas lighting, mobbing, lying, prompting costly activities, shaming, vilifying, playing the victim role, evasion, initiating confusion, and seduction (Paulhus & Williams, 2002; Braiker, 2004; Packard, 2007). Unfortunately, these, and many other coercive communication techniques, are unrecognisable to students’ and educators’ untrained ears and eyes. A lack of systems thinking in the higher education curriculum prevents students’ and academics’ understanding and discovering patterns and interrelationships, in terms of intimidation and manipulation practice.\n\nAuthors (Senge, 1990; Ross & Jon, 2015) have emphasised a lack of systems thinking in educational practice and theories. Ross and Jon (2015, p. 10) define systems thinking as “a set of synergistic analytic skills used to improve the capability of identifying and understanding systems, predicting their behaviours, and devising modifications to them in order to produce desired effects; these skills work together as a system”.\n\nResearchers (Senge, 1990; Ross & Jon, 2015; Richmond, 1994) point out that education entities are responsible for the empowerment of systems thinking. This can lead to enhancing academics’ insights so that they generate a model for detecting and preventing subtle psychological attacks. By applying systems thinking, it is possible to comprehend the multifaceted behaviours of coercive control as interdisciplinary systems, with the purpose of predicting behaviour and adjusting their outcomes (Richmond, 1994; Sweeney & Sterman, 2000; Cabrera et al., 2008; Plate, 2010; Ross & Jon, 2015).\n\nBasically, a system thinking skill set could help educators to view the system of coercive control from an intuitive domain and in a holistic way (Bertalanffy, 1967, as cited in Mononen, 2017; Mella, 2012). System thinking skills may empower academics to see an overall structure and the patterns and cycles of an intimidation or manipulation system (for example, the political, economic, cultural, social, community, administration, management, policy, and psychological domain) (Thaler & Sunstein, 2008; Packard, 2007). Systems thinking skills can be observed as a subset of critical thinking skills, and the implementation of system-oriented instruction should be placed within the context of long-term educational goals (Plate & Monroe, 2014).\n\nScientific evidence is lacking about the effectiveness of interventions to prevent non-violent behaviour (Adorno & Becker, 1999; Ttofi & Farrington, 2011) and to deal with intimidation bullying and harassment (Low et al., 2013). Researchers (Scott, 2014; Menesini & Salmivalli, 2017) highlight the use of disciplinary practices, raising peer awareness and peer group pressure, promoting anti-bullying standards, and having discussions in the classrooms. There has been minimal research on bullying climates in college environments, or on the efforts that institutions are employing to reduce intimidation instances on their own campus (Salmivalli & Voeten, 2004; Scott, 2014).\n\nSentse, Kiuru, Veenstra and Salmivalli (2014) propose a social network approach for addressing bullying among adolescents, pointing out that bullying is a group process and, consequently, context dependent. Mononen (2017) supports a variety of measures, such as life skills, social development, mentoring, neighbourhood, conflict management, and schools-based anti-bullying prevention programmes. Scott (2014) proposes anti-bullying efforts, such as an institutional-wide effort to provide proactive anti-bullying intervention programmes. The researchers, Ttofi and Farrington (2011), support the assignment of peers, as educators and awareness trainers, as a crucial intervention to inhibit bulling, as well the implementation of an anti-bullying policy, although they caution that having any kind of policy in place might not be enough.\n\nAccording to Van Dijk (2006, p. 371), one of the best ways to detect and resist hidden control attempts is to acquire specific knowledge about the interests of the manipulator and a general knowledge about psychological manipulation. It is in the best interest of dominant groups to make sure that relevant and potentially critical general knowledge is not acquired by those who are being controlled, or that only partial, misguided, or biased knowledge is allowed for distribution (Thaler & Sunstein, 2008; Van Dijk, 2006; Packard, 2007; Tepper et al., 2008; Veenstra et al., 2013).\n\nBecause of a lack of coordination in prevention and regulation procedures at higher education institutions, the proposed measures and proactive programming methods (Mononen, 2017; Scott, 2014) are mostly ineffective across higher education institutions. Thus, there are no widely accepted measures to deal with intimidation and coercive control in educational contexts, as students are unable to engage on social media with others in a frank or intimate way (Adorno, 1998). As such, we need a model that is intended to function as a structural framework that can create a fruitful atmosphere for coordinated actions against intimidation and manipulative behaviour in higher education institutions and across society.\n\n\nA Model of Coercive Control (MCC)\n\nThe aim of the study was to develop a model that could produce an awareness impact on decision makers, researchers, and students and that could assist in detecting, preventing, and rectifying coercive intimidation and influence as a contagious social problem. Consequently, the MCC was created: it was based on systems thinking theoretical perspectives (e.g., Richmond, 1994; Sweeney & Sterman, 2000; Cabrera et al., 2008; Ross & Jon, 2015) and on research on intimidation and manipulation (Dahlberg & Etienne, 2002; Anderson & Bushman, 2002; Musselman et al., 2005; Tepper et al., 2008; Mononen, 2017; Karim & Duchcherer, 2014), in order to clarify the process of coercive control, with specific application to higher educational contexts.\n\nResearch findings (Vanlneveld, Cook, Kane, & King, 1996; Salmivalli & Voeten, 2004; Buss, 2005; Cialdini, 2006; Packard, 2007) provide a solid conceptual background in creating the model. There are hardly any fundamental insights into a detailed procedure regarding the process and flow of coercive intimidation and manipulation, or any discussions about a structural approach (Van Dijk, 1996, 2006), and this may be caused by a lack of systems thinking on intimidation and coercive influence. This will be discussed in the following sections.\n\nThe authors developed the model, assuming that it might illuminate major hidden components, might describe the process and the flow of the coercive impact as an advanced feature of coercive intimidation, and might provide a vision into specific and general knowledge of manipulative techniques (Paulhus et al., 2002; Cialdini, 2006; Van Dijk, 2006; Packard, 2007; Veenstra et al., 2013) that were targeted towards students and educators. The model consists of six major components that need to be considered when analysing the coercive control process and its flow:\n\n1. The control/funding entity (decision makers, spokesmen, informers, controllers, intimidators);\n\n2. The targeted individual (personality, history, experience, emotions, vulnerability).\n\n3. The programme (programmers, designers, technical experts).\n\n4. Network environments (family network, social media network, institutional networks, and informal networks).\n\n5. Channels of communication (the means/technology to transmit messages and receive feedback; social media, technical unit operators, and managers); and\n\n6. An outcome report (the human resources and the technology to operate recording and producing a report).\n\nThe first element presents the control unit, organised by the founding or control body, which appoints representatives and allocates specific roles (researcher(s), information providers, operational manipulators, a spokesman, and the programme designer) to implement, monitor, and control the manipulation process.\n\nThe second element is a targeted individual. The individual has specific cognitive, personal characteristics, experience, capabilities, and vulnerabilities.\n\nThe third element is a software programme, with predefined programming messages designed with a purpose to change personality and behaviour of the target individual, based on research and the hidden aim of the coercive process.\n\nThe fourth element is the network environment(s), which consists of the individual networks and discourses.\n\nThe fifth element is a communication channel: in other words, the technology used to transmit the programming sequences, in the form of a human assistant and/or a technological transmitter.\n\nThe sixth element is the outcome, in the form of an assessment report.\n\nThe model can be expanded, and additional components and networks can be added. The components of the MCC model are presented in the form of successive stages connected with the flow of arrows that indicate two directional control feedbacks (see Figure 1).\n\nThe following stages have been identified within the coercive process, which are interwoven with the main components and the control feedback:\n\nStage I – Forming and organising the control/funding unit (organising funds, allocating roles and activities, designing a feedback control mechanism, providing strategies for monitoring and controlling, clarifying informing procedures, managing the unit);\n\nStage II – Performing research (choosing an individual and his or her network, gathering personal history and experience data, assessing his or her personality traits, cognition, emotions, and vulnerability scope);\n\nStage III – The software programme development (algorithm design, coding, getting the technology ready to transmit programming sequences);\n\nStage IV – Operational planning (creating the action plan and the map of activities, predicting obstacles, and developing measures to rectify, selecting appropriate technology, checking the feedback system);\n\nStage V – Implementation of the programme (applying technology and off-line human agents to transmit programming messages, monitoring performance and repetitions in a calculated time sequence, recording observed data); and\n\nStage VI – Outcome assessment (applying methods and technology to analyse recorded data, creating a report for further programme improvement) (see Figure 1).\n\nThe first stage introduces the core control entity that begins the coercive impact process, targets the individual and networks, and prepares data for the programme design. The control entity has been empowered with systems thinking (Ross & Jon, 2015; Plate, 2010; Richmond, 1994). Since the motivation to design the programme is pragmatic, the programme design (in stage III) depends on research data of the target person and his or her networks, done in stage II (see Figure 1).\n\nThe six stages enlighten the social coercive process flow, implicitly encompassing sub-systems, namely academic networks, family networks, and community networks, which are all inter-connected coercive influence are intrinsically interrelated with two-directional flows of information feedback, silently transferred through discourse interactions, from the individual to the control centre and back to the individual.\n\nEach component in the model is integrated within the proceeding and successive stages and works in a synergy. The model provides a general conceptual framework, without specifications of techniques that are already available in the research (Paulhus et al., 2002; Mucchielli, 2003; Braiker, 2004; Packard, 2007; Stanciugelu, 2010).\n\nEach building block of the model contributes to a sustainable manipulative programming context and must be understood quite well by policy makers and targeted individuals, in order to prevent further coercive influence. Additionally, all stakeholders (e.g. academics, students, decision makers, funders, industry partners, researchers) have their place in different stages in the model, playing different roles.\n\nThe structure and the flow of the model are drawn from theoretical perspectives that support the crucial value of critical thinking (Veenstra et al., 2013; Paulhus et al.,2002; Cialdini, 2006) and systems thinking (Ross & Jon, 2015; Sweeney & Sterman, 2000; Plate, 2010). The model is flexible, and it can help to predict the flow of control and inhibiting factors.\n\nThe model contains pre-programmed discourse messages and non-verbal reinforcement stimuli to which the individual is exposed on a subconscious level (Wodak, 1987; Van Dijk, 2006; Rojo & Van Dijk, 1997), synchronised with action and discourse techniques of other members in the network (Braiker, 2004; Baldry, Farrington, & Sorrentino, 2015; Matz, Kosinski, Nave, & Stillwell, 2017). The programming procedure inspires many discourses, interactions, and activities, via a recurrence method, and students internalise its impact and, subsequently, influence others in the network through modelling (Paulhus et al., 2002; Cialdini, 2006; Van Dijk, 2006; Menesini & Salmivalli, 2017). The individual will spontaneously follow the stimuli of programmed manipulative messages that are transmitted through expert reinforcement technology, discourses, and imposed social interactions within their own networks (Althusser, 1970).\n\nConsequently, the networks of manipulative influence are constantly expanding because of the repetitive process emerging into changed personalities, into unhealthy relationships, and, finally, into destructive conflicts within networks that negatively influence students’ academic achievements (Cialdini, 2006; Thaler & Sunstein, 2008; Al-Raqqad et al., 2017). Relevant social and institutional environments are adjusted to maintain the continuous flow of manipulative silent messages. The MCC model presents the flow of coercive techniques that influence the sub-conscious mind of youth and adults and that direct and monitor their actions, led by the programme and the control centre.\n\nProgramming sequences are transferred to the target before the actual realisation of a manipulative procedure, with the purpose to keep the constant flow of stimuli and to prevent distractions. Psychological targeting makes it possible to influence the behaviour of groups of people by tailoring persuasive appeals to the psychological needs of targeted audiences (Thaler & Sunstein, 2008; Baldry et al., 2015; Matz et al., 2017). Youths and students are usually individually targeted, but they are not aware of the coercive control procedure, and they cannot detect and avert further influences, because of multiple reasons, e.g., a lack of general and specific knowledge and a lack of critical thinking and information filtering (Mucchielli, 2003; Musselman et al., 2005; Van Dijk, 2006; Packard, 2007; Veenstra et al., 2013).\n\nThe whole coercive control cycle is invisible; it is an imitation of the communication processes at work in the brain and it is interpreted by target people as a product of their own internal decision-making process (Van Dijk, 2006). Thus, an individual is exposed to a conventional realm of creation, impossible to comprehend because of learning processes and consequent personality changes (Bell, 1948; Paulhus et al., 2002) within a confined psychological space and predefined activities and interactions that are out of her or his control.\n\nThe control unit usually emerges from a higher societal system, and it is concealed under an array of interests (for example, economic, scientific, medical, political, and educational) with the aim to change and influence covertly the individual and their networks. The coercive control process is executed and monitored by its creators and the outcome is predicted, based on input stimuli. Depending on the type of programme, its influence can be enormously destructive, since it affects and spreads to other individuals and networks with behaviour, attitude, and belief system changes and can hardly be reversed.\n\nThe model is based on the systems thinking foundations, the practicalities of research findings, the prevalent techniques, tactics, causes, types, and measures, and the technology means (Salmivalli & Voeten, 2004; Tepper et al., 2008; Scott, 2014). Through the model, individuals can modify their personal capabilities, traits, beliefs and attitudes, based on controlled manipulated interactions within the family, educational institutions, and the societal and work environments (Baldry et al., 2015).\n\nTherefore, the MCC model reflects an innovative design of the coercive process, based on current theory and practice (Mucchielli, 2003; Braiker, 2004; Tepper et al., 2008; Stanciugelu, 2010) and is an effort that may contribute to a workable solution that decision makers may consider to challenge proactively this invisible social threat. Furthermore, the model has a fundamental structure, elements, flow, dynamics, and a feedback control that reflect a generic pathway. An individual receives a regular dose of manipulative messages through human and technological means and channels, particularly via whispering, discourse, and auditory messaging (Van Dijk, 2006; Packard, 2007).\n\nIn summary, the model presents a basic coercive-control life cycle, and the individual should be responsible for self-monitoring, observing, and informing decision bodies at educational settings. Institutions are responsible for initiating awareness programmes, developing training material and ensuring human resources to maintain and monitor non-violent incidences for students and academics.\n\n\nDiscussion\n\nThis article argues that a lack of coercive intimidation and control awareness within the current curriculum and a lack of institutional supremacy to reverse fragmented knowledge on coercive manipulation at universities are warning signs of inadequate students’ and academics’ security and well-being.\n\nFurthermore, in this article, multiple intimidation and manipulation issues were discussed (for example, Vanlneveld et al., 1996; Buss, 2005; Salmivalli & Voeten, 2004; Van Noorden et al., 2016), and a systems thinking theoretical framework was introduced as a basis for derivation of the model, that may serve as a critical aid to students, academics, and decision makers in higher education institutions. Thus, the model was derived through a combination of theoretical, practical, and reflective experiences as an attempt to understand the impact of the multiple-faceted nature of the coercive process (see Figure 1).\n\nThe documentary analysis indicates a dynamic intersection of numerous factors of intimidation, bullying and harassment, and hidden manipulation tactics and techniques (Mucchielli, 2003; Braiker, 2004; Cialdini, 2006; Tepper et al., 2008) that work at a sub-conscious level (Van Dijk, 2006, p. 361; Stanciugelu, 2010). Systems thinking skills can be observed as a subset of critical thinking skills, and implementation of system-oriented instruction should be placed within the context of long-term educational goals (Plate & Monroe, 2014).\n\nThe documentary analysis indicates the importance of knowledge about psychological manipulation: “the recipients lack the specific knowledge that might be used to resist manipulation” (Wodak, 1987, as cited in Van Dijk, 2006, p. 360; Packard, 2007) and forms of hidden attacks (Van Dijk,2006, p. 371), but there are no detailed investigations into how manipulation is carried out, how to determine the victim’s vulnerability to hidden control, and what are means of early detection. Research findings (e.g., Mononen, 2017; Scott, 2014) highlight multiplicity of preventive measures, but there is no agreement on what preventive and corrective measures could be used in higher education.\n\nThe first research question seeks to determine the following: “What are the major components of a model of coercive control in higher educational contexts (MCC)?” The model introduces six components: the control/funding entity; the targeted individual; the programme; the network environments; channels of communication; and outcome assessment report.\n\nAlthough the six components are depicted as separate entities, they interact synergistically, in the sense that every variable may influence and guide the others within the identified stages. Consequently, components of the model are interconnected between stages: these stages include a feedback control; monitoring; and the channelling of subconscious influence that the targeted person cannot detect or understand because manipulative messages pass the control of the conscious mind.\n\nThe second research question seeks to determine the following: “What are the stages of coercive control at institutions of higher education?” Researchers are aware of manipulation techniques, tricks, channels, discourses, and types (Mucchielli, 2003; Van Dijk, 1996, 2006; Stanciugelu, 2010), but few findings were recorded, regarding clear methods of infiltrations, and there is no clarity regarding a structural, organised framework or about the flow of influence, control feedbacks, and the diversities of human or technical resources. Subsequently, the MCC model reveals some aspects, especially the organised and clearly phased process that can influence the success of manipulation with no visible traces. The following six stages were derived: stage I – forming and organising the control unit; stage II – performing research; stage III – the software programme development; stage IV – operational planning; stage V – implementation of the programme; and stage VI – outcomes assessment.\n\nCoercive infiltrations are not included in the curriculum at higher education institutions, but researchers are puzzled as to why students are reluctant to exercise self-control (Duckworth, White, Matteucci, Shearer, & Gross, 2011) that reflects diminished defensive strength against intimidation (Foucault, 1977; Menesini & Salmivalli, 2017). With adequate proactive programmes (Scott, 2014), critical knowledge acquisition and exchange (Veenstra et al., 2013), and awareness training, as preventive measures against organised non-violent coercive intrusions (Ttofi & Voeten, 2011), security preparedness may flourish within educational domains.\n\nHigher education institutions, with their resources and opportunities, play a vital role in training, supporting, and coordinating actions to detect, prevent, and remedy organised manipulative attacks (Salmivalli et al., 2004; Scott, 2014). Academics and students can benefit from the MCC model due to its novelty, its provision of a detailed structure about the flow of control of the process, and its knowledge about coercive infiltrations and their contagious nature and invisibility.\n\n\nConclusion\n\nThe article explored theoretical and research viewpoints on intimidation and the dynamics of coercive manipulation in higher education, and it described numerous facets and measures to detect, prevent, and rectify these social threats. In summary, an in-depth analysis of literature, current practices at universities, and the derivation of the conceptual model reveal the following tentative conclusions:\n\n• Multiple measures undertaken by educational institutions, for instance, school policies, anti-bullying awareness programmes, and regulatory, security and government measures cannot guarantee, prevent or rectify the impact of coercive intimidation and infiltration tactics and its psychological harm, mal-development or deprivation (Salmivalli & Voeten, 2004; Beale & Hall, 2007).\n\n• There is an urgent need for a curriculum change that may serve as a point of departure, so that pre-college learners are better informed to develop altruistic and humanitarian values and are capacitated to question critically policies, government, and the media.\n\n• Adequate awareness and training programmes in relation to coercive infiltration are missing at higher education institutions. Students and staff members lack a satisfactory knowledge base that enables them to create counterarguments and to understand norms, values, and ideologies, and they have ambiguous critical-thinking capacities that cannot counteract the persuasive arguments advanced by groups and organisations.\n\n• Systems thinking and system thinkers are rare in educational environments (Senge, 1990; Richmond, 1994; Ross & Jon, 2015), and this prevents a deeper understanding of coercive, manipulative subsystems and it impedes students and educators from adequately analysing this social problem.\n\n• Security education and training and the awareness of technology are urgently needed in educational contexts (Martinez & Schilling, 2010).\n\n• Higher education institutions should focus on strengthening critical thinking and encouraging inquiring minds, scepticism, and non-conforming behaviour (Mucchielli, 2003; Veenstra et al., 2013). Since our students are lacking these basics, the problem of counter-discourses is less serious for the manipulators, and, therefore, students are more vulnerable and less resistant to manipulation (Van Dijk, 2006).\n\nIn the light of the discussion in this article, it can be concluded that the youth are the most vulnerable because of their inexperience, lack of knowledge, and their inadequate awareness measures and appropriate training. Skinner (1955–56) suggests that we must continue to develop laws and systems of government that will prevent the governing body, from using its power to enslave others.\n\nIn summary, the following suggestions are offered:\n\nResearchers have recognised the current fragmented approach to interpretations of intimidation and manipulative practice, as well as a deficiency in the availability of an appropriate theoretical framework, which may influence inadequate applications of preventive and remedial measures. Thus, researchers agree with Adorno (1998) who argues that “… it is still an open question whether the individual, enlightened and made critically aware … might not still in his behaviour be open to manipulation and control in some way …”. He hopes that “education and enlightenment can still manage a little something …”\n\nThe design of a novel MCC model, which is applicable for higher education institutions, and which is based on a solid theoretical framework, with the purpose to clarify the complex social problem of coercive control, may be regarded as the originality and the value of this research. Additionally, this paper aims to inspire researchers to undertake further research on this topic, specifically in response to the in-depth analysis of crucial coercive control components and stages.\n\nThe limitations can result from a lack of technological specifications of the model. The conclusions of this study should be cautiously applied in higher educational contexts because of the necessity for practical investigations in real environments. Thus, the dynamics of, and the components contained in, the structure of the proposed model need further analysis, including clear assessment procedures, to identify early signs of organised coercive infiltration.\n\n\nData availability\n\nFigshare: A model of coercive control in higher education: a qualitative study. https://doi.org/10.25399/UnisaData.20120627.v3. (Jakovljevic, 2022)\n\nThis project contains the following underlying data:\n\n• Prof Marija Jakovljevic Data file 1_ Methods of data managements processing and analysis_14 June 2022.pdf\n\n• Prof Marija Jakovljevic Data file 2_Additional textual data extracts 14 June 2022.pdf\n\n• Prof. Marija Jakovljevic Data file 3_Databases searches 14 06 2022.pdf\n\nThis project contains the following extended data:\n\n- Prof Marija Jakovljevic data set-SRQR checklist.pdf\n\n- Prof M Jakovljevic ETHICAL Clearance UNISA.pdf\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\nAdorno T: Education after Auschwitz. Critical models: interventions and catchwords. Pickford HW, editor.New York:Columbia University Press;1998; 191–204.\n\nAdorno T, Becker H: Education for maturity and responsibility. Translated by Robert French, Jem Thomas, and Dorothee Weymann. Hist. Hum. Sci. 1999; 12(3): 21–34. Publisher Full Text\n\nAlvesson M, Sköldberg K: Reflexive methodology—new vistas for qualitative research. London:Sage;2000. Reference Source\n\nAlthusser L: Ideology and ideological state apparatuses. Lenin and Philosophy and Other Essays. New York:Monthly Review Press;1970; 85–126. 2001.\n\nAl-Raqqad HK, Al-Bourini ES, Talahin FM, et al.: The Impact of school bullying on students’ academic achievement from teachers’ point of view. Int. Educ. Stud. 2017; 10(6): 44–50. Publisher Full Text\n\nAnderson CA, Bushman BJ: Human aggression. Ann. Rev. Psychol. 2002; 53: 27–51. 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Publisher Full Text\n\nBuss S: Valuing autonomy and respecting persons: manipulation, seduction, and the basis of moral constraints. Ethics. 2005; 115: 195–235. Publisher Full Text\n\nBurrowes RJ: The psychology of ideology and religion.2016Reference Source\n\nCabrera D, Colosi L & Lobdell C : Systems thinking. Evaluation and program planning. Evaluation and Program Planning. 2008; 31(3): 299–310. Reference Source\n\nCAIR: Position paper on intimidation and harassment.1996.Reference Source\n\nCialdini RB: Influence: The psychology of persuasion. Collins Business Essentials;2006. Revised Edition. ISBN: 006124189X ISBN13: 9780061241895 ASIN: 006124189X. Reference Source\n\nDahlberg LL, Etienne GK: Violence a global public health problem: Version of the introduction to the World Report on Violence & Health (WHO). Geneva:World Health Organization Division of Violence Prevention;2002.\n\nDenzin N, Lincoln Y: Handbook of qualitative research. 2nd ed.Thousand Oaks:Sage Publications;2000.\n\nDilmac B: Psychological needs as a predictor of cyber bullying: a preliminary report on college student. Educ. Sci.: Theory Pract. 2009; 9(3): 1307–1325.\n\nDuckworth AL, White RE, Matteucci AJ, et al.: A stitch in time: Strategic self-control in high school and college students. J. Educ. Psychol. 2011; 108(3): 329–341. Free Full Text\n\nFarmer PE, Nizeye B, Stula S, et al.: Structural violence and clinical medicine. PLoS Med. 2006; 3(10): 1686–1691. Publisher Full Text\n\nFook J: The reflective researcher: social workers. Theories of Practice research, editor.Sydney:Allen & Unwin;1996.\n\nFook J: Critical social work. London:Sage;2002.\n\nFook J: Critical reflection and transformative possibilities.Davies L, Leonard P, editors. Social Work in a Corporate Era: Practices of Power and Resistance. Aldershot:Ashgate;2004; pp. 16–30.\n\nFoucault M: Discipline and punish: the birth of the prison. New York:Vintage;1977.\n\nGaltung J: Cultural violence. J. Peace Res. 1990; 27(3): 291–305. Publisher Full Text\n\nJakovljevic M: A model of coercive control in higher education: a qualitative study. University of South Africa;2022. Dataset. Publisher Full Text\n\nKarim S, Duchcherer M: Intimidation and harassment in residency: a review of the literature and results of the 2012. Can. Med. Educ. J. 2014; 5(1): e50–e57. Free Full Text\n\nKoch T, Harrrington A: Reconceptualizing rigour: the case for reflexivity. J. Adv. Nurs. 1998; 28(4): 882–890. Publisher Full Text\n\nKothari CR: Research methodology: Methods and techniques. New Delhi:New Age International Publishers;2004.\n\nLow S, Polanin JR, Espelage DL: The role of social networks in physical and relational aggression among young adolescents. J. Youth Adolesc. 2013; 42: 1078–1089. PubMed Abstract | Publisher Full Text\n\nMahon JE: A Definition of deceiving. Int. J. Appl. Philos. 2007; 21: 181–194. Publisher Full Text\n\nMatz SC, Kosinski M, Nave G, et al.: Psychological targeting as an effective approach to digital mass persuasion. Proc. Natl. Acad. Sci. U. S. A. 2017; 114(48): 12714–12719. PubMed Abstract | Publisher Full Text\n\nMeriläinen M, Sinkkonen H, Puhakka H, et al.: Bullying and inappropriate behaviour among faculty personnel. Policy Futures in Education. 2016; 14(6): 617–634. Publisher Full Text\n\nMartinez M, Schilling S: Using technology to engage and educate youth. New Dir. Stud. Dev. 2010; 2010: 51–61. Publisher Full Text\n\nMella P: Systems thinking. Intelligence in action. Springer Science & Business Media;2012. 978-88-470-2564-6. Publisher Full Text\n\nMenesini E, Salmivalli C: Bullying in schools: the state of knowledge and effective interventions. Psychol. Health Med. 2017; 22(1): 240–253. PubMed Abstract | Publisher Full Text Reference Source\n\nMitnick KD, Simon WL: The Art of deception: controlling the human element of security. Hoboken, NJ, USA:Wiley;2003. Reference Source\n\nMononen L: Systems thinking and its contribution to understanding future designer thinking. Proceedings of the 12th European Academy of Design Conference. 2017; (pp. S4529–S4538). Design Journal, 20, Suppl. 1. Taylor & Francis. Publisher Full Text\n\nMorley C: Chapter 14: Critical reflection as a research methodology. Knowing Differently: Arts-Based and Collaborative Research. Liamputtong P, Rumbold J, editors.Nova Science publishers. Inc.;2008; pp. 265–280. 97-8-I-60456-378-8.\n\nMucchielli A: The art of influence. the analysis of the techniques of manipulation. Iasi:Polirom Publishing;2003.\n\nMusselman L, McRae H, Reznick R, et al.: You learn better under the gun: intimidation and harassment in surgical education. Med. Educ. 2005; 39: 926–934. PubMed Abstract | Publisher Full Text\n\nNoble H, Smith J: Qualitative data analysis: A practical example. Evid. Based Nurs. 2013; 17(1).Reference Source\n\nOjo MA: Religious reportage in the Contemporary Nigerian. Press. Paper read at Religion and Media in Nigeria, at SOAS, London. 1999.\n\nOlweus D: Aggression in the schools: Bullies and whipping boys. New York, NY:Hemisphere Publishing;1978.\n\nOlweus D: Bullying at school: What we know and what we can do. Oxford:Blackwell;1993.\n\nPackard V: The hidden persuaders. Ig Publishing;2007. 9780978843106. 097884310X. Review paper by David Edwards.\n\nPaulhus D, Williams KM: The dark triad of personality: Narcissism, Machiavellianism and psychopathy. J. Res. Pers. 2002; 36(6): 556–563.\n\nPlate R: Assessing individuals’ understanding of nonlinear causal structures in complex systems. Syst. Dyn. Rev. 2010; 26(1): 19–33. Publisher Full Text\n\nPlate R, Monroe M: A structure for assessing systems thinking. The Creative Learning Exchange. 2014; 23(1): 1–12.Reference Source\n\nThe Progressive Teachers Union of Zimbabwe [PTUZ] Report: Political violence and intimidation against teachers in Zimbabwe. The Research and Advocacy Unit [RAU]. 2002.Reference Source\n\nRichmond B: Systems dynamics/systems thinking let’s just get on with it. Proceedings of Conference International Systems Dynamics. Sterling, Scotland:1994.\n\nRitchie J, Lewis J, Elam G: Designing and selecting samples.Ritchie J, Lewis J, editors. Qualitative research practice. A guide for social science students and researchers. Thousand Oaks, CA:Sage;2003; (pp.77–108). Reference Source\n\nRojo LM, Van Dijk TA: There was a problem, and it was solved: legitimating the expulsion of illegal migrants in Spanish parliamentary discourse. Discourse Soc. 1997; 8(4): 523–566. Publisher Full Text\n\nRoss DA, Jon W: A definition of systems thinking: a systems approach. Procedia Comput. Sci. 2015; 44: 669–678. Publisher Full Text Reference Source\n\nRosaldo R: Subjectivity in social analysis: from culture and truth.Seidm S, editor. The postmodern turn: new perspectives on social theory. Cambridge University Press;1994; pp.171–183.\n\nRossiter A: Discourse analysis in critical social work: from apology to question. Crit. Soc. Work. 2005; 6(1).\n\nSalmivalli C, Voeten M: Connections between attitudes, group norms, and behaviours associated with bullying in schools. Int. J. Behav. Dev. 2004; 28: 246–258. Publisher Full Text Reference Source\n\nScott J: Bullying and cyber-bullying in Higher Education: Current institutional practice and prevention. Educational Leadership and Policy Studies. The Graduate Faculty of the University of Kansas;2014.Reference Source\n\nSenge P: The fifth discipline, the art, and practice of the learning organization. New York, NY:Doubleday/Currency;1990.\n\nSentse M, Scholte R, Salmivalli C, et al.: Person-group dissimilarity in involvement in bullying and its relation with social status. J. Abnorm. Child Psychol. 2007; 35: 1009–1019. Publisher Full Text\n\nSentse M, Kiuru N, Veenstra R, et al.: A social network approach to the interplay between adolescents’ bullying and likeability over time. J. Youth Adolesc. 2014; 43(9): 1409–1420. PubMed Abstract | Publisher Full Text\n\nSimon GK: In sheep's clothing: understanding and dealing with manipulative people.1996. 978-1-935166-30-6.\n\nSkinner BF: Section of psychology: The control of human behaviour. The New York Academy of Sciences. 1955; 17(7): 547–551. Series II. Publisher Full Text\n\nSmyth J, Shocklock G: Behind the cleansing socially critical research accounts.Smyth J, Shocklock G, editors. Being reflexive in critical educational and social research. London:Folmer Press;1998; pp. 1–12.\n\nSnyder H: Literature review as a research methodology: An overview and guidelines. J. Bus. Res. 2019; 104: 333–339. Publisher Full Text\n\nStanciugelu S: The logic of manipulation: 33 techniques of Romanian political manipulation. C. H. Beck;2010. 978-973-115-736-8.\n\nStuckey HL: The second step in data analysis: Coding qualitative research data. J. Soc. Health Diabetes. 2015; 3(1).\n\nStrauss A, Corbin J: Basics of qualitative research: Techniques and procedures for developing grounded theory. Thousand Oaks, CA:Sage;1998.\n\nSweeney LB, Sterman JD: Bathtub dynamics: initial results of a systems thinking inventory. Syst. Dyn. Rev. 2000; 16(4): 249–286. Publisher Full Text Reference Source\n\nTesch R: Qualitative research: Analysis types and software tools. New York, NY:Falmer;1990.\n\nVanlneveld C, Cook D, Kane S, et al.: Discrimination and abuse during internal medicine residency. J. Gen. Intern. Med. 1996; 11: 401–405. PubMed Abstract | Publisher Full Text\n\nVeenstra R, Dijkstra JK, Steglich C, et al.: Network-behaviour dynamics. J. Res. Adolesc. 2013; 23: 399–412. Publisher Full Text\n\nTepper BJ, Henle CA, Lambert LS, et al.: Abusive supervision and subordinates’ organization deviance. J. Appl. Psychol. 2008; 93(4): 721–732. PubMed Abstract | Publisher Full Text Reference Source\n\nThaler RH, Sunstein CR: Nudge: improving decisions about health, wealth, and happiness. New Haven & London:Yale University Press;2008. 978-0-300-12223-7.Reference Source\n\nTtofi MM, Farrington DP: Effectiveness of school-based programs to reduce bullying: A systematic and meta-analytic review. J. Exp. Criminol. 2011; 7: 27–56. Publisher Full Text\n\nUN: Convention on the Rights of the Child.1989. Reference SourceReference Source\n\nVan Dijk TA: Discourse, power, and access.Caldas-Coulthard CR, Coulthard M, editors. Texts and Practices: Readings in Critical Discourse Analysis. London:Routledge;1996; pp. 84–104.\n\nVan Dijk TA: Discourse and manipulation. SAGE Publications (London, Thousand Oaks, CA & New Delhi). Discourse Soc. 2006; 17(2): 359–383. Publisher Full Text Reference Source\n\nVan Noorden THJ, Bukowski WM, Haselager GJT, et al.: Disentangling the frequency and severity of bullying and victimization in the association with empathy. Soc. Dev. 2016; 25: 176–192. Publisher Full Text\n\nVitoroulis I, Vaillancourt T: Meta-analytic results of ethnic group differences in peer victimization. Aggress. Behav. 2015; 41: 149–170. Publisher Full Text\n\nWodak R: And where is the Lebanon? A socio-psycholinguistic investigation of comprehension and intelligibility of news. Text.1987; 7(4): 377–410."
}
|
[
{
"id": "146319",
"date": "08 Aug 2022",
"name": "Nataša Simić",
"expertise": [
"Reviewer Expertise Psychology",
"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 paper examines a very relevant topic that has not been studied enough in the context of HE and offers a model of psychological coercive control as a result of the qualitative analyses. Full potential of the paper to impact scientific public would be fulfilled if the authors improve clarity of some parts.\nIn the introductory part many different concepts are used. It would be good to briefly provide at least some common definitions, especially for systems thinking that has an important place in the aims but was not clearly defined before. Research questions could be better linked to the aims.\nIf the themes were presented in a form of table or diagram, it would be easier for readers to follow. Later referral to these themes in the Results section would also help.\nIn the methodological sections it is all described, but when one reads the results, it is sometimes not clear enough what is the result of the literature review, what of the reflective synthesis, what of the documentary analysis and if there were some additional types of analysis. Finally, it would be good if the authors explain in more details what they base their model on.\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Partly\n\nAre sufficient details of methods and analysis provided to allow replication by others? Partly\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nNot applicable\n\nAre all the source data underlying the results available to ensure full reproducibility? Partly\n\nAre the conclusions drawn adequately supported by the results? Yes",
"responses": [
{
"c_id": "8632",
"date": "16 Aug 2022",
"name": "Maria Jakovljevic",
"role": "Author Response",
"response": "Dear reviewer, the following are answers to your report: 1. Provide common definitions 1a. A lack of systems thinking among students exabits persistent errors in their understanding of multifaceted systems and educators are seeking to develop systems thinking skills. Systems thinking involves the ability to represent and assess dynamics of basic building blocks of complex systems, discover processes, the flow, recognize and challenge the boundaries (Sweeney & Sterman, 2000). 1b. Coercive control includes control of another person’s behavior, emotions and thinking, by applying force, threats or triggering fear. 2. Research questions could be better linked to the aims The objectives of the study provided a basis to conceptualize the following research questions: 3. Present themes in a table The selected written data sources were critically analyzed and reflectively synthetized into nine final themes, see Table 1. Table 1 Major themes Themes ... The themes were evaluated as the most relevant in explaining the phenomena of psychological manipulation in higher education environments and as a basis for the model design (see Table 1). 4. Explain in more detail what they base their model on A reflective and critical synthesis was conducted that revealed key elements of the model drawn from major themes on crucial concepts of coercive manipulation, e.g., intimidation techniques, systems thinking, manipulative channels, forms of manipulation, which served as a basis for the model (see Table 1). Furthermore, the MCC was based on systems thinking theoretical perspectives (e.g., Richmond, 1994; Sweeney et al., 2000; Cabrera et al., 2008; Ross et al., 2015) and on research on intimidation and manipulation (Dahlberg et al., 2002; Anderson et al., 2002; Musselman et al., 2005; Tepper et al., 2008; Mononen, 2017; Karim et al., 2014), in order to clarify the process of coercive control, with specific application to higher educational contexts."
},
{
"c_id": "9045",
"date": "04 Jan 2023",
"name": "Maria Jakovljevic",
"role": "Author Response",
"response": "Responses to Reviewer 1 The paper examines a very relevant topic that has not been studied enough in the context of HE and offers a model of psychological coercive control as a result of the qualitative analyses. Full potential of the paper to impact scientific public would be fulfilled if the authors improve clarity of some parts. In the introductory part many different concepts are used. It would be good to briefly provide at least some common definitions, especially for systems thinking that has an important place in the aims but was not clearly defined before. Research questions could be better linked to the aims. This may also be caused by a lack of systems thinking skills as a subset of critical thinking skills that may enable students and educators to detect hidden signs of coercive control, understand the act of persuasion, and take defending steps in their network (Thaler & Sunstein, 2008; Packard, 2007). (3) to create a model of psychological coercive control with unique components and stages that will inspire academics to take collaborative steps to respond to intimidation and manipulative influence at their institutions. The emerging questions set in this paper are: Research Question 2: What are the stages of coercive control at institutions of higher education? If the themes were presented in a form of table or diagram, it would be easier for readers to follow. Later referral to these themes in the Results section would also help. The selected written data sources were critically analyzed and reflectively synthetized into eight final themes and they were evaluated as the most relevant in explaining the phenomena of psychological manipulation in higher education environments and as a basis for the model design ( Smyth & Shocklock, 1998; Alvesson & Sköldberg, 2000; Ritchie, et al., 2003). See Table 1. Table 1: The results of documentary analysis of textual data The major themes Bullying and harassment are prevalent forms of intimidation. Antibullying programs are widely applied for concurring bullying and intimidation. A lack of standardization and common regulations on anti-bullying measures. Communication acts of dominance, positioning, and language discourses encourage psychological manipulation. A systematic theory, a framework, the structure, and processes of manipulation are absent in higher education. Predominant subconscious and deceptive nature of psychological manipulation. Education of maturity, courage, resolution, and systematic reflection as preventive measures. Enabling a comprehension of coercive control through teaching systems thinking The major themes emerged from the documentary analysis of textual data on bullying, harassment, and other forms of intimidation that indicate the existence of various antibullying programs, and measures for prevention and rehabilitation, however, there are no standardized procedures, regulations, the teaching of systems thinking, a systematic theory, a model, the structure and processes across academic communities that may be due to subconscious and deceptive nature of psychological manipulation (see Table 1). The documentary analysis indicates the importance of knowledge about psychological manipulation: “the recipients lack the specific knowledge that might be used to resist manipulation” ( Wodak, 1987, as cited in Van Dijk, 2006, p. 360; Packard, 2007) and forms of hidden attacks ( Van Dijk,2006, p. 371), but there are no detailed investigations into how manipulation is carried out, how to determine the victim’s vulnerability to hidden control, and what are means of early detection (see Table 1). The second research question seeks to determine the following: “What are the stages of coercive control at institutions of higher education?” Researchers are aware of manipulation techniques, tricks, channels, discourses, and types ( Mucchielli, 2003; Van Dijk, 1996, 2006; Stanciugelu, 2010), but few findings were recorded, regarding clear methods of infiltrations, and there is no clarity regarding a structural, organised framework or about the flow of influence, control feedbacks, and the diversities of human or technical resources (see Table 1). In the methodological sections it is all described, but when one reads the results, it is sometimes not clear enough what is the result of the literature review, what of the reflective synthesis, what of the documentary analysis and if there were some additional types of analysis. Finally, it would be good if the authors explain in more details what they base their model on. As a result of the documentary analysis and reflective synthesis, there was no evidence of a model of coercive control in HE, meaning that there is a gap in research in this area (see Table 1). This has inspired the researchers of this study to use their reflections, previous work on coercive control, and their creative insight into manipulative processes on a subconscious level that resulted in a design of a model that may stimulate research interest in this area and promote awareness of this widely spread phenomena in HE."
}
]
},
{
"id": "153267",
"date": "02 Nov 2022",
"name": "Mirna Marković",
"expertise": [
"Reviewer Expertise Organizational Psychology",
"Higher Education Management",
"Cognitive 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\nI strongly welcome the research initiative of the authors and I believe this article has a high degree of interest considering the lack of qualitative research in the field of psychological coercive influence in higher education. It is relevant considering the current knowledge in this field and can be characterized by precision in the use of conceptual and terminological apparatus and clarity and stylistic precision of linguistic expression.\nIn order for the article to be more scientifically sound and aligned with the needs of practice, I point out some aspects that the authors could further address. These suggested changes are not expected to significantly change either the concept or the structure of the work - they are aimed at enriching part of its content, which the authors, given their knowledge of the field, will probably easily recognize.\nAlthough the presented model of coercive control (MCC) in higher educational contexts is based on a reflexive analysis and a critical synthesis of relevant scientific literature, the impression is that it is mostly oriented towards students - there are very few references to academic staff. I suggest that the authors comment on this fact in the paper in order to highlight their position on the (under)capacitation of scientific research in this area or offer some other comment regarding this observation.\n\nSince the proposed model has development potential, it would be useful to offer a more precise description of the elements and dynamics of the phases assumed by the model. I appreciate that, among other things, it would be useful to refer to some concrete examples from the university environment, on the basis of which it would be possible to more easily examine the assumptions of the model and form some new constructive, reflective questions.\n\nAlthough the conclusions given at the end of the article are very transparent and clear, one gets the impression that a more specific review of the model itself is missing.\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Partly\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nNot applicable\n\nAre all the source data underlying the results available to ensure full reproducibility? Partly\n\nAre the conclusions drawn adequately supported by the results? Yes",
"responses": [
{
"c_id": "9044",
"date": "04 Jan 2023",
"name": "Maria Jakovljevic",
"role": "Author Response",
"response": "Responses to Reviewer 2 I strongly welcome the research initiative of the authors and I believe this article has a high degree of interest considering the lack of qualitative research in the field of psychological coercive influence in higher education. It is relevant considering the current knowledge in this field and can be characterized by precision in the use of conceptual and terminological apparatus and clarity and stylistic precision of linguistic expression. In order for the article to be more scientifically sound and aligned with the needs of practice, I point out some aspects that the authors could further address. These suggested changes are not expected to significantly change either the concept or the structure of the work - they are aimed at enriching part of its content, which the authors, given their knowledge of the field, will probably easily recognize. Although the presented model of coercive control (MCC) in higher educational contexts is based on a reflexive analysis and a critical synthesis of relevant scientific literature, the impression is that it is mostly oriented towards students - there are very few references to academic staff. I suggest that the authors comment on this fact in the paper in order to highlight their position on the (under)capacitation of scientific research in this area or offer some other comment regarding this observation. The references to academic staff were added throughout the article where it was necessary. Since the proposed model has development potential, it would be useful to offer a more precise description of the elements and dynamics of the phases assumed by the model. I appreciate that, among other things, it would be useful to refer to some concrete examples from the university environment, on the basis of which it would be possible to more easily examine the assumptions of the model and form some new constructive, reflective questions. The components of the MCC Model At this level initiating coercion makes it easy for others to implement coercion consciously or unconsciously. Those appointed will typically be made comfortable in their roles and typically receive incentives for facilitating coercions. These characteristics will be identified by the control unit and categorized as strengths and weaknesses. Weaknesses are exploited and strengths avoided. The third element is a software programme, with predefined programming messages designed with the purpose to change the personality and behavior of the academics and youths as target individuals, based on research and the hidden aim of the coercive process. Coercive behavior is automated and becomes implementable and acceptable as an integral part of the control body strategies. With the software in place, cohesion can always be made more sophisticated and successful with little or no intervention from the original software developers or the control body. Here technology can be used to communicate information regarding what it means to be, for example, incompetent based on the observed behavior of the victim. Networks and discourses tend to legitimize actions seen in elements 1-3. They tend to confuse the victims on the origin and find themselves getting intimidation by multiple sources. Victims, without knowledge of systems theory, may blame themselves instead of the perpetrators. The stages of MCC model The following scenario illustrates the first stage in a form of an off-line programming event in a library. The student comes to a library intending to learn, where he/she hears the message spontaneously transferred, ‘I cannot learn…I am tired…no reward for learning…’ Similar messages are repeatedly transferred at the same place after a certain time when the memory about previous similar incidents has declined. The student slowly gets aversion to this learning context but cannot explain why. The student is closely monitored to detect the pattern of movements, behavior, the language discourse, and with pre-programmed messages, his/her behavior and willingness to study gradually are changing in a hidden way. The method of psychological manipulation with software programs (stage III), and other information communication technologies is out of the scope of this paper. Dynamics within the MCC A symbiosis exists between the components, the flow, and the stages of the model, in the sense that the process of manipulation is accumulated, new habits are formed and the sub-conscious acceptance of a coercive environment as it is natural. Students and educators gradually lose critical thinking due to a learning process of manipulative assimilation as victims learn to keep it secret and they are enforced to follow messages without questioning as there is no time for self-reflection. Pre-programmed sequences of other members in the academic network are adjusted to fulfil the coercive program of a target individual. The dynamics of pre-programmed messages is a repetitive process that influences a further change of behavior, emotions, and cognition of members, that contagiously spread to multiple academic situations, to encompass more members and other networks in the society. Summary of the MCC Model HE environments have been targeted, due to the vulnerability of youths and educators, caused by their human capital potential, a lack of knowledge of social coercion control, and a lack of this sub-discipline in the HE curriculum. Although the conclusions given at the end of the article are very transparent and clear, one gets the impression that a more specific review of the model itself is missing. Conclusion Thus, the youth and educators are vulnerable, and they need to know their rights and where to seek them. The MCC model with its complex structure, components, stages, process, flow, and dynamics provided a deeper insight into psychological manipulation in HE. However, the baseline of this study should be used for empirical research and the practical applications of the MCC model in different HE contexts. Thus, the MCC model provides a building block for further examination of coercive control in HE environments."
}
]
}
] | 1
|
https://f1000research.com/articles/11-880
|
https://f1000research.com/articles/12-2/v1
|
03 Jan 23
|
{
"type": "Research Article",
"title": "Mental health interventions targeting children and young people: A mapping review of interventions, follow-up, and evidence gaps",
"authors": [
"Astrid Dahlgren",
"Ingrid Borren",
"Brynhildur Axelsdottir",
"Mari Elvsåshagen",
"Karianne Hammerstrøm Nilsen",
"Ingrid Borren",
"Brynhildur Axelsdottir",
"Mari Elvsåshagen",
"Karianne Hammerstrøm Nilsen"
],
"abstract": "Background: Young people with mental illness should be offered evidence-based treatments. In 2018, we developed a national evidence portal in Norway providing mental health professionals and others with living evidence summaries. This immense work has been an important contribution to mental health care in Norway but is also a rich data source for exploring the characteristics and evidence gaps of the existing research internationally. At the time of this study, eight overviews of systematic reviews (OoOs) had been published. These addressed treatments for attention deficit / hyperactivity disorder (ADHD), anxiety, depression, bipolar disorder, psychosis, obsessive compulsive disorder (OCD), self-harm and trauma/ post-traumatic stress disorder. The objective of this study was to do a secondary analysis of this evidence to describe the state-of-the art in this field, and to map:\ntreatments evaluated for each patient group and the longest time of follow-up treatment comparisons evaluated for more than one patient group\nMethods: We performed a mapping review of the eight OoOs. Data extraction was performed by one author and double-checked by another. All data was entered into Excel. Findings were visualized in descriptive tables and using Sunburst-diagrams. We used statistical thresholds to determine the size of effect and report the associated certainty. Results: We identified 200 treatment comparisons including a wide variety of interventions. Some mental illnesses are treated mostly with pharmacological or combination therapies and others solely with psychological or psychosocial treatments or with more diversity. The evidence supporting most treatments is of low to very low certainty. Ten percent of the comparisons included follow-up assessments beyond 12 months. Cognitive behavioural therapy, dialectic behavioural therapy, physical activity and mindfulness interventions were effective across populations. Conclusions: The evidence supporting treatment of mental illness in young people has important limitations. Future research efforts should address these evidence gaps.",
"keywords": [
"mental health",
"children",
"youth",
"interventions",
"mapping review",
"evidence gaps"
],
"content": "Introduction\n\nActing early by providing children and youth with evidence-based treatments for mental health problems may prevent chronic illness and deterioration of other health outcomes and quality of life.1 Attention-deficit/hyperactivity disorder (ADHD), behavioural disorders and internalising disorders such as anxiety and depression are common disorders diagnosed in childhood and adolescence.2 In general, as many as one in four are reported to experience a mental disorder, and according to the WHO mental health disorders are among the leading causes of disability worldwide.1,2\n\nInterventions targeting mental illness encompass a range of treatments including (but not restricted to) psychological therapies, pharmacological therapies, peer-supported interventions, educational interventions, physical activity, nutritional interventions and alternative therapies such as acupuncture and yoga. Traditionally, treatments for mental health problems have taken a diagnostic approach, however a new school of thought argue for a transdiagnostic approach that cuts across traditional diagnostic boundaries.3\n\nChildren and young people should be offered evidence-based treatments. Summarised evidence is a necessity for good quality health care and should inform decision-making about treatment choices.4,5 However, there is an abundance of systematic reviews published every year and many of these are not of satisfactory quality.6 Evidence summaries such as overviews of systematic reviews are therefore important decision-making tools for patients, professionals and policy makers.7\n\nIn 2018 we developed a national evidence portal (see here) in Norway hosting living evidence summaries of systematic reviews evaluating the effects of interventions for children and young people with mental illness.8,9 The evidence portal relies on best practice review methodology and was inspired by other international evidence portals targeting mainly adults and somatic illness.10 At the time the present study was conducted, eight overviews of systematic reviews (here termed ‘OoOs’) had undergone peer-review and were published in the evidence portal. In an OoO, all available systematic reviews meeting explicit inclusion and exclusion criteria are summarized and quality assessed. Sometimes OoOs are also called umbrella reviews. Each of the OoOs addressed a specific patient group, and summarized the effects of treatments for children and young people with ADHD, anxiety, depression, bipolar disorder, psychosis (including schizophrenia), obsessive-compulsive disorder (OCD) self-harm and trauma (including post-traumatic stress disorder, PTSD) respectively.11–18\n\nThis immense work has been an important contribution to mental health care in Norway and has been integrated in the Norwegian national guidelines but is also a rich data source for exploring the characteristics and evidence gaps of the existing research. Furthermore, it can also be used for exploring the effects of treatments across patient groups. This is valuable input into the contemporary debate on transdiagnostic approaches, and the potential value of some treatments to be effective for several mental illnesses.3\n\n\nObjectives\n\nThe purpose of this study was to do a secondary analysis of the eight evidence summaries included in the evidence portal and to describe the state- of- the art of the existing research in this field. Specifically, we aimed to:\n\n(1) map all treatments evaluated for each patient group and the longest time of follow-up included in these assessments\n\n(2) map treatment comparisons evaluated across groups and to create an overview of the effects of these including the certainty of the evidence (using the Grading of Recommendations Assessment, Development and Evaluation framework – GRADE - on the primary outcome for each patient group\n\nTo our knowledge, no mapping review describing the diversity of interventions for treating mental illness in children and young people exist.\n\n\nMethods\n\nThere is no gold standard for conducting mapping reviews, however a common set of guiding rules have been suggested.19,20 In general, mapping reviews provide an overview of the literature by describing and organising the research literature according to characteristics such as interventions, intervention components or population. Mapping reviews are also used for identifying evidence gaps, and to inspire and guide new research initiatives.19,20 The protocol for this review has been published.21\n\nThere is an abundance of systematic reviews summarising the effects of interventions for child and young people’s mental health. We have in the above-mentioned previous work completed eight OoOs including systematic reviews evaluating all interventions targeting children and adolescents (0-18 years) with; ADHD, anxiety, depression, bipolar disorder, psychosis (including schizophrenia), OCD, self-harm and trauma (including PTSD).11–18 The analysis reported on in this paper is in its entirety based on these eight OoOs and represents a secondary analysis of what we consider to be complete summaries of the effectiveness of interventions reported on in high quality systematic reviews.\n\nOur analysis is restricted to the inclusion and exclusion criteria of the eight OoOs. The protocols for these overviews have been published in PROSPERO (ADHD, CRD42020159885; Anxiety, CRD42020159884; Bipolar, CRD42020176356; Depression, CRD42020159883; Psychosis, CRD42020212244; Self-harm, CRD42019117942; Trauma CRD42019120078; OCD, CRD42020221081), however we briefly summarise the approach we applied here.\n\nAll OoOs were conducted adhering to the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) checklist and according to “best practice”-principles of review methods.10 The search strategies for the OoOs were largely based on the database for systematic reviews IN SUM (indexing publications from all major databases. The search strategy applied by the IN SUM database can be accessed here). All systematic reviews indexed in IN SUM were considered for inclusion. We also hand searched national databases of evidence-based guidelines in Sweden, Denmark and the UK (NICE). The flow chart describing each step of the inclusion and exclusion of publications can be seen in Figure 1.\n\nPICO, patient intervention comparison outcome; OCD, obsessive compulsive disorder; ADHD, attention deficit/hyperactivity disorder; OoO, overviews of systematic reviews.\n\nTo be included in one of the OoOs, a review had to meet a set of minimum criteria (4 out of 5) for being considered “systematic”, set by DARE.22\n\n1. Clear inclusion and exclusion criteria\n\n2. A comprehensive search strategy\n\n3. A compilation of results from included studies, or\n\n4. Quality assessment of included studies.\n\n5. Sufficiently reported details about the individual included studies\n\nFurthermore, all interventions intended to improve an aspect of mental health for children and adolescents under the age of 18 were included.\n\nSome of the OoOs included both preventive and treatment interventions. For this mapping review, we only considered interventions targeting children and adolescents with a clinical diagnosis of mental illness or otherwise judged by the systematic review authors to have moderate to serious symptomatology. The reason for this is that not all the OoOs included preventive studies and would thus render this secondary analysis incomplete.\n\nThe OoOs excluded studies where pharmacological interventions were not compared to active interventions, thus placebo-controlled studies were excluded. Furthermore, studies evaluating mental health interventions targeting people where the primary health concern was somatic (e.g., patients with asthma and co-occurring anxiety) were excluded.\n\nThe overviews are “living reviews”, and so for the sake of this mapping review we based our data collection on the latest updated version of each overview respectively, with the most recent of these updates including evidence published in April 2021 or later. The data collection for this mapping review took place between September 2021 and December 2022.\n\nWe mapped all interventions evaluated. In the results section, we present these findings in descriptive charts (Sunburst-diagrams) for each patient group respectively. We also mapped the number of treatment comparisons evaluated and the longest time of follow-up included in these assessments (for any outcome).\n\nTo create a mega-map of intervention effects across patient groups we also extracted data on the primary outcome for each intervention, and the judgement of certainty for this outcome.23,24 All data were entered into Excel. The primary outcome was conceptualised as overall symptomatology associated with the relevant condition, such as anxiety symptoms for those with anxiety and depression symptoms for those with depression. In cases where the same outcome was assessed using different outcome measures, we extracted the outcome with the longest follow up and highest certainty.23 If a review did not report findings on overall symptoms, we reported other symptoms as proxy, for example inattention in treatment of ADHD when total symptoms were not available.\n\nSince this is a mapping review, we based our analysis on the effect-sizes and judgements of certainty as they were reported in the OoO. Judgements about certainty included in the OoO were made using the GRADE-criteria.23,24 GRADE (Grading of Recommendations, Assessment, Development and Evaluations) is a transparent and widely adapted tool for developing and presenting summaries of evidence.23 Using this framework, the evidence is judged to have high, moderate, low or very low certainty by considering the following criteria: risk of bias, imprecision, inconsistency, indirectness, publication bias, magnitude of effect, dose-response gradient and residual confounding.\n\nWe extracted the effect sizes and categorized these “small”, “moderate” or “large” based on statistical rule-of-thumb judgements (see Table 1).10,24\n\nWhat is considered “a meaningful effect” depends on the individual patient and context. Thus, we made no attempt to consider the clinical importance of these results as this is best judged by those delivering and receiving treatments. In some cases, it was difficult to judge the size of the effect as the results were not reported using standardized or relative effect estimates. In these circumstances we used the systematic review authors’ own judgements and have annotated these in the results table. If no judgements were made by the review authors, we marked this as “effective” without making judgements about the size of effect. Trivial or small not statistically significant differences in effect was coded as “little or no difference” according to the GRADE-recommendations.25\n\nAll data extraction was done by one author and double checked by another co-author. Any difference in opinion between these were discussed with a third co-author.\n\n\nResults\n\nWe reviewed 116 systematic reviews included in the eight OoOs (see Figure 1). As the OoOs are living documents and regularly updated, we have included a list of the systematic reviews included in the summaries at the time data was extracted for this study – see Underlying data.30\n\nThe evidence underlying treatment options of mental illness for children and young people includes 200 treatment comparisons evaluating the primary outcome for each specific diagnostic group. See Underlying data29 for more information.\n\nOverall, 49.5% of the treatment comparisons were non-pharmacological interventions, 36% included pharmacological or nutrition/supplemental interventions, and 14.5% were combination treatments including both pharmacological and non-pharmacological treatments.\n\nThe number of treatment comparisons varied greatly across the patient groups; from 69 comparisons for ADHD to 7 for OCD (see Table 2).\n\nADHD, attention deficit/hyperactivity disorder; OCD, obsessive compulsive disorder; PTSD, post-traumatic stress disorder.\n\nInterventions by treatment categories are displayed in Sunburst-diagrams by patient groups (see Figures 2–10). The evidence informing ADHD-treatment includes a range of psychological, psychosocial, dietary, systemic, physical activity, skills training and pharmaceutical interventions. About half of the ADHD-interventions are pharmacological or combination therapies including medication.\n\nCBT, cognitive behavioural therapy; DBT, dialectic behavioural therapy; NRI, norepinephrine reuptake inhibitors.\n\nCBT, cognitive behavioural therapy; MBSR, mindfulness based stress reduction.\n\nCBT, cognitive behavioural therapy; DBT, dialectic behavioural therapy.\n\nCBT, cognitive behavioural therapy; DBT, dialectic behavioural therapy; ECT, electroconvulsive therapy; rTMS, repetitive transcranial magnetic stimulation; MBSR, mindfulness based stress reduction.\n\nCBT, cognitive behavioural therapy; iCBT, internet-based cognitive behavioral therapy; ERP, exposure and response prevention; SSRI, selective serotonin reuptake inhibitors.\n\nCBT, cognitive behavioural therapy.\n\nCAT, cognitive analytic therapy; CBT, cognitive behavioral therapy; DBT, dialectic behavioural therapy; MBT, mentalization-based therapy.\n\nCBT, cognitive behavioural therapy; EFT, emotion-focused therapy; EMDR, eye movement desensitisation and reprocessing; TF-CBT, trauma focused cognitive behavioral therapy.\n\nEMDR, eye movement desensitisation and reprocessing; CBT, cognitive behavioural therapy; TF-CBT, trauma focused cognitive behavioral therapy.\n\nMost treatment evaluations for depression are non-pharmacological and include different types of psychological therapies, physical activity, art therapy and medical treatments such as electroconvulsive therapy (ECT) and repetitive transcranial magnetic stimulation (rTMS). The evidence also includes pharmacological interventions including use of serotonin-norepinephrine reuptake inhibitors and selective serotonin reuptake inhibitors (SNRIs/SSRIs) in combination with cognitive behavioural therapy (CBT) or SSRI used alone.\n\nThe evidence for treating OCD represents less diversity, and most interventions are CBT-based, either used alone or in combination with medications.\n\nTreatment evaluations for bipolar disorder and psychosis include mainly pharmacological treatments, either as direct comparisons or in combination with psychological treatments or other medications (bipolar disorder). Psychological treatments used for psychosis include psychoeducation, computer-based cognitive training, CBT and group- and family therapy. For bipolar disorder, evaluations also include psychoeducation, CBT, interpersonal therapy, Dialectic behavioural therapy (DBT) and family therapy.\n\nSelf-harm and trauma/PTSD-interventions are all psychological, psychosocial, or other therapeutic interventions such as psychoeducation. Several organisational or systemic treatments used for preventing suicide or reoccurrence of self-harm have also been evaluated, including interventions such as the use of “emergency green card” (self-admittance to specialist health care).\n\nThe longest follow-up for any outcome could be found for anxiety (6 years), followed by ADHD and depression (3 years), bipolar, self-harm and PTSD (2 years), trauma (1 year) and OCD (6 months).\n\nOverall, approximately 40% of the treatment comparisons included only short-term follow-up (< 3 months). A total of 10% of the treatment comparisons included follow-up beyond 12 months (see Table 3).\n\nFollow-up times by diagnosis and category can be seen in Table 4. Overall, it can be observed that for most patient groups, treatment comparisons of pharmacological treatment included very short follow-up.\n\nADHD, attention deficit/hyperactivity disorder; OCD, obsessive compulsive disorder; PTSD, post-traumatic stress disorder.\n\nThere was a large diversity of treatment comparisons evaluated. We identified 24 unique groups of treatment comparisons evaluated for more than one patient group. These can be seen in Table 5.\n\nADHD, attention deficit/hyperactivity disorder; OCD, obsessive compulsive disorder; PTSD, post-traumatic stress disorder; CBT, cognitive behavioral therapy; DBT, dialectic behavioural therapy; TAU, treatment as usual; TF-CBT, trauma focused cognitive behavioral therapy; EMDR, eye movement desensitisation and reprocessing.\n\n* Several outcomes available, outcomes with larger effect and certainty included here.\n\n** Difficult to assess size of effect, review authors report the intervention as “effective”.\n\n*** Several outcomes available, outcomes with larger effect and certainty included here. Pharmacological treatment included stimulants (ADHD), SSRI (depression) and TCA (anxiety).\n\n**** Several outcomes available, outcomes with larger effect and certainty included here. Pharmacological treatment included antipsychotics (psychosis) and SSRI (anxiety). Pharmacological treatment for bipolar and ADHD not specified.\n\nMost treatment evaluations were associated with low or very low certainty. The certainty was higher for treatment evaluations including CBT, which also demonstrated moderate to large effects compared to treatment as usual across different patient groups. DBT compared to treatment as usual has been evaluated for depression and self-harm with low to moderate effect sizes on primary symptoms (low certainty). Physical activity is found to have a moderate effect on primary symptoms of ADHD and depression (low certainty). Mindfulness interventions also result in small but beneficial effects compared to treatment as usual (TAU) for anxiety and depression (moderate to low certainty).\n\nLittle or no important difference in effect was found when comparing psychological treatments with pharmacological treatments for depression and OCD, however a small but beneficial effect in favour of psychological treatment was found for anxiety (all low certainty). Combination therapy of CBT and pharmaceutical treatments was found to have a moderate effect on anxiety, but there was little or no difference for ADHD when compared to psychological treatment alone. The certainty of the evidence for combination therapy compared to pharmacological therapy alone, was very low for three out of four patient groups evaluating this treatment comparison (anxiety, bipolar and psychosis). For ADHD, combination treatment was found to produce large effects on the primary outcome, although with low certainty.\n\nTreatment comparisons including psychoeducation (vs TAU), family therapy (vs TAU), psychodynamic (vs TAU), Eye Movement Desensitization and Reprocessing (EMDR) (vs TAU), massage therapy (vs TAU) is very uncertain across all patient groups evaluated.\n\n\nDiscussion\n\nOur analysis is based on eight recent overviews of systematic reviews summarising the evidence reported in 116 systematic reviews.11–18 This analysis includes only direct comparisons of treatments reported on in high quality systematic reviews. To our knowledge this is the first mapping review of its kind supporting treatment of child and youth mental illness, and our findings is of great relevance to clinicians, funders of new research initiatives and researchers with a blueprint for planning future research activities. Furthermore, acknowledging research uncertainties is an important step-stone in good patient care.\n\nOne limitation to our work is that research is accumulating rapidly in this field, and that while this report is being written, new evidence may have emerged. However, considering the substantial evidence gaps we identified we are confident that our conclusions may continue to be valid some time to come. Another limitation may be that our analysis is based on reviews, and that the results are impacted by the methodological choices and reporting by the authors of the individual reviews included in each OoO, but also by the inclusion criteria by the OoO. For example, this analysis does not include placebo-controlled pharmaceutical studies or comparisons of preventive treatments. As such, we would like to emphasise that there is a large body of evidence evaluating these treatment comparisons and that the results of these and the corresponding evidence gaps should be considered together with our analysis. Network analyses of indirect comparisons are an important contribution to this evidence base.26 Furthermore, our analysis is based on treatments evaluated in specific patient groups. Other reviews have summarised the effects of interventions in diverse patient groups (not restricted to diagnostic criteria). Thus, implementation of findings coming out of our analysis should take into consideration the findings of such transdiagnostic reviews.\n\nWe identified 200 treatment comparisons. The most striking finding of this review is the short follow-up times for most treatment comparisons. This is the case for pharmaceutical treatments in particular. Approximately 40% of all treatment comparisons included only short-term follow-up (<3 months), with only 10% of the treatment comparisons including a follow-up beyond 12 months. Thus, there is great uncertainty associated with the long-term effects of mental health treatments for children and young people.\n\nWe found large diversity in treatments evaluated. Our descriptive analysis shows that the largest diversity can be found for ADHD and less diversity was found for OCD. Furthermore, from the research that has been conducted so far it can be deducted that the etiology and assumed relevant interventions are understood differently depending on the diagnosis. Some conditions are, for instance, addressed mostly by combination treatments or pharmaceutical treatments alone whereas others are treated with psychological or psychosocial interventions. For example, most treatments for bipolar disorder were pharmacological, whereas treatments for children with PTSD/trauma were all psychological or psychosocial therapies. Treatments for ADHD and psychosis include a combination of both pharmaceutical and psychological or psychosocial treatments. This is an important contribution to the epistemology of mental health, and the understandings of what may reduce or increase mental health depending on the symptoms we identify (diagnosis).\n\nThe certainty of most treatment outcomes is low or very low. However, there are treatments with promising effects, which also seem to be effective across patient groups. CBT demonstrates moderate to large effects (compared to treatment as usual) across four patient groups. DBT therapy, physical activity and mindfulness interventions also result in small to moderate beneficial effects compared to TAU (moderate to low certainty). Combination therapies including CBT and pharmacological treatments was either found to have similar effects on primary symptoms compared to either CBT or pharmacological treatment alone, or to be more effective (low certainty).\n\nPractitioners and decision-makers should be aware of important research uncertainties and make these explicit in communication with patients and when developing guidelines. Our report may be an important platform for doing so.\n\nFor many treatment evaluations the evidence is limited. Nevertheless, some treatments have shown convincing treatment effects and should be considered as first choice when treating children and young people with mental illness. Some treatments identified in our review may be well documented for use in adults and may therefore also be applied to younger people. Although this may be theoretically sound, the transferability to and the effectiveness for younger people is unclear and should be addressed. There is therefore a need for more high-quality research on the effectiveness of treatments with uncertain effects on children and young people. Future research efforts should also include long-term follow up assessments, as this is fundamental to patient safety.\n\nWe have made no judgements about the clinical importance of the effect-sizes we mapped in this review, as this is a decision which is best left up to health professionals and their patients. Furthermore, we did not review or make any judgements about which outcomes were evaluated in the treatment comparisons we mapped. For pragmatic reasons, our analysis depended on the primary outcome (change in symptoms) for each diagnostic group using statistical rule of thumb thresholds. It is important to emphasise that any changes in symptomatology should be supplemented with measuring other relevant outcomes. The opinions of patients and health professionals about the relevance of outcomes may differ from those of researchers. Thus, future studies should include the opinions of patients and should consider any established core outcome sets.27,28\n\n\nConclusions\n\nOur analysis provides essential information through a mapping review about the state-of the art of the existing evidence.\n\nWe identified a wide range of treatments evaluated for use in children and young people, including psychological, social, dietary, physical activity, skills training and pharmacological interventions. Based on this review, it can be observed that some mental illnesses are treated mostly with pharmaceutical or combination therapies and others solely with psychological or psychosocial treatments or with more diversity. With few exceptions, the evidence supporting most treatments is of low to very low certainty. CBT demonstrates moderate to large effects across four patient groups with moderate to low certainty. DBT, physical activity and mindfulness interventions also demonstrate small to moderate beneficial effects compared to TAU (moderate to low certainty). Most concerningly, we observed that the majority of the existing treatment evaluations included very short follow-up measurements. Health professionals and policy makers should consider these uncertainties when communicating with patients. Future research efforts should target important research uncertainties identified in this review and plan for longer follow-up times.\n\n\nData availability\n\nZenodo: Included comparisons_mapping review_2022. https://doi.org/10.5281/zenodo.6948764.29\n\nThis project contains the following underlying data:\n\n- Treatment comparisons mapping review_2022_english.xlsx (comparisons included in the analysis of this paper).\n\nZenodo: Supporting materials_mapping review_2022. https://doi.org/10.5281/zenodo.6815213.30\n\nThis project contains the following underlying data:\n\n- Appendix_mapping_june 2022.docx (systematic reviews included in the OoOs).\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).",
"appendix": "References\n\nMurray CAL: World Health Report 2002: Reducing Risks, Promoting Healthy Life. Geneva, Switzerland:World Health Organization;2002.\n\nMerikangas KR, Nakamura EF, Kessler RC: Epidemiology of mental disorders in children and adolescents. Dialogues Clin. Neurosci. 2009; 11(1): 7–20. PubMed Abstract | Publisher Full Text | Free Full Text\n\nDalgleish T, Black M, Johnston D, et al.: Transdiagnostic approaches to mental health problems: Current status and future directions. J. Consult. Clin. Psychol. 2020; 88(3): 179–195. PubMed Abstract | Publisher Full Text\n\nDawes M, Summerskill W, Glasziou P, et al.: Sicily statement on evidence-based practice. BMC Med. Educ. 2005 Jan 5; 5(1): 1.Publisher Full Text Reference Source\n\nAmerican Psychological Association: Evidence-Based Practice in Psychology American Psychological Association.2005.Reference Source\n\nChalmers I, Bracken M, Djulbegovic B, et al.: How to increase value and reduce waste when research priorities are set. Lancet. 2014; 383(9912): 156–165. PubMed Abstract | Publisher Full Text\n\nStraus S, Haynes RB: Managing evidence-based knowledge: the need for reliable, relevant and readable resources. Can. Med. Assoc. J. 2009; 180(9): 942–945. PubMed Abstract | Publisher Full Text\n\nRBUP East and South: Tiltakshandboka: et oppslagsverk over forskning på barn og unges psykiske helse: RBUP East and South.Reference Source\n\nAustvoll-Dahlgren AB, Hammerstrøm KT, Kjøbli J, editors: Enabling informed treatment choices: development of an evidence portal for children and young people's mental health in Norway (oral-presentation).Abstracts of the 25th Cochrane Colloquium; 2018; Edinburgh, UK.\n\nHiggins J, Thomas J, Chandler J, et al.: Cochrane Handbook for Systematic Reviews of Interventions version 6.2 (updated February 2021).Reference Source\n\nDahlgren A, Morken I, Nøvik T, et al.: Kunnskapsoppsummering: effekt av tiltak for bipolar lidelse hos barn og unge. Tiltakshåndboka: oppsummert forskning om effekt av tiltak for barn og unges psykiske helse.2020 [cited 2021 15.11].\n\nAxelsdottir B, Borren I, Dahlgren A, et al.: Kunnskapsoppsummering: effekt av tiltak for angstlidelser hos barn og unge. Tiltakshåndboka: oppsummert forskning om effekt av tiltak for barn og unges psykiske helse.December 2021.Reference Source\n\nAxelsdottir B, Eidet L, Dahlgren A, et al.: Kunnskapsoppsummering: effekt av tiltak for depresjon hos barn og unge. Tiltakshåndboka: oppsummert forskning om effekt av tiltak for barn og unges psykiske helse.2021 15.11.2022.Reference Source\n\nDahlgren A, Morken I, Karlsen K, et al.: Kunnskapsoppsummering: effekt av tiltak for psykoselidelser hos barn og unge. Tiltakshåndboka: oppsummert forskning om effekt av tiltak for barn og unges psykiske helse.2020 15.11.2021.Reference Source\n\nMorken I, Dahlgren A, Lunde I, et al.: Kunnskapsoppsummering: effekt av tiltak for selvskading og selvmordsatferd hos barn og unge. Tiltakshåndboka: oppsummert forskning om effekt av tiltak for barn og unges psykiske helse.2020 15.11.2021; 2020.Reference Source\n\nBorren I, Eidet L, Bræin M, et al.: Kunnskapsoppsummering: effekt av tiltak for traumerelaterte lidelser hos barn og unge. Tiltakshåndboka: oppsummert forskning om effekt av tiltak for barn og unges psykiske helse.2021 15.11.2021; 2021.Reference Source\n\nDahlgren A, Axelsdottir B, Borren I, et al.: Kunnskapsoppsummering: effekt av tiltak for tvangslidelser hos barn og unge Tiltakshåndboka: oppsummert forskning om effekt av tiltak for barn og unges psykiske helse.2020.Reference Source\n\nElvsåshagen M, Dahlgren A, Eidet L, et al.: Kunnskapsoppsummering: effekt av tiltak for ADHD og andre hyperkinetiske forstyrrelser hos barn og unge. Tiltakshåndboka: oppsummert forskning om effekt av tiltak for barn og unges psykiske helse.2020 15.11.2021.Reference Source\n\nGrant MJ, Booth A: A typology of reviews: an analysis of 14 review types and associated methodologies. Health Inf. Libr. J. 2009; 26(2): 91–108. PubMed Abstract | Publisher Full Text\n\nArksey H, O'Malley L: Scoping studies: towards a methodological framework. Int. J. Soc. Res. Methodol. 2005; 8: 19–32. Publisher Full Text\n\nDahlgren A, Axelsdottir B, Borren I, et al.: Protocol: A mega-map of mental health interventions targeting children and young people. Center for Open Science;2021-09-13.Reference Source\n\nCentre For Rreviews and Dissemination: The database of Abstracts of reviews of effects (DARE) University of York.2002. [Accessed: 02.09.2021].Reference Source\n\nGuyatt GH, Oxman AD, Schunemann HJ, et al.: GRADE guidelines: A new series of articles in the Journal of Clinical Epidemiology. J. Clin. Epidemiol. 2011; 64(4): 380–382. Publisher Full Text\n\nSchünemann H, Brożek J, Guyatt G, et al.: GRADE Handbook.2013. Accessed: 02.09.2021].Reference Source\n\nSantesso N, Glenton C, Dahm P, et al.: GRADE guidelines 26: informative statements to communicate the findings of systematic reviews of interventions. J. Clin. Epidemiol. 2020; 119: 126–135. Publisher Full Text\n\nCorrell CU, Cortese S, Croatto G, et al.: Efficacy and acceptability of pharmacological, psychosocial, and brain stimulation interventions in children and adolescents with mental disorders: an umbrella review. World Psychiatry. 2021; 20(2): 244–275. PubMed Abstract | Publisher Full Text\n\nWilliamson PR, Altman DG, Blazeby JM, et al.: Developing core outcome sets for clinical trials: issues to consider. Trials. 2012; 13(1): 132. PubMed Abstract | Publisher Full Text\n\nJames Lind Alliance: Priority setting partnerships.Reference Source\n\nDahlgren A: Included comparisons_mapping review_2022. Zenodo. Dataset. 2022. Publisher Full Text\n\nDahlgren A: Supporting materials_mapping review_2022. Zenodo. Dataset. 2022. Publisher Full Text"
}
|
[
{
"id": "199270",
"date": "06 Sep 2023",
"name": "Hiran Thabrew",
"expertise": [
"Reviewer Expertise Child and adolescent mental health clinician and researcher"
],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThank you for the opportunity to review this interesting paper summarizing results of a mapping review of interventions for common child and adolescent mental health problems.\nThe strengths of this paper are:\nThe use of a comprehensive national database of evidence.\n\nThe inclusion of most common child and adolescent mental health problems.\n\nThe clearly presented information about how the study was conceptualized, designed and implemented, including adequate supplementary material with which the review may be replicated.\n\nClear tables and figures to summarize results.\n\nInclusion of length of follow-up data to highlight the short-term nature of most studies.\n\nAreas for improvement are as follows:\nMy main concern about this paper is the limitation of included studies to those which had used an active comparator (not placebo). This is despite the source OoOs including studies with any type of comparator:\nStatement from mapping review: \"The OoOs excluded studies where pharmacological interventions were not compared to active interventions, thus placebo-controlled studies were excluded.\"\nStatement from protocol of ADHD review: \"Comparator(s)/control: Any interventions or treatment as usual (TAU).\"\nI'm not sure why placebo controlled studies were excluded from the mapping review, and as a result, its findings are technically interesting, but of limited clinical benefit. A key example is how methlyphenidate pharmacotherapy for ADHD is not explicitly mentioned in Table 5, even though it is the mainstay of treatment for this condition worldwide. Greater explanation of this issue (or, dare I ask for it, a reworking of the analysis including all types of studies) would be useful.\n\nUsing colors more consistently in the sunburst diagrams (Figures 2-10) to make it easier for the reader to compare the weight of evidence for each type of therapy across different conditions (e.g., medication is blue in one diagram, yellow in another).\n\nPlease explain the colors and question marks used in Table 5.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Partly\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes",
"responses": []
}
] | 1
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https://f1000research.com/articles/12-2
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https://f1000research.com/articles/11-1296/v1
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11 Nov 22
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{
"type": "Research Article",
"title": "Health literacy on COVID-19 and COVID-19 vaccinations in Indonesia",
"authors": [
"Viskasari P. Kalanjati",
"Nurina Hasanatuludhhiyah",
"Annette d'Arqom",
"Azlin Muhammad",
"Ancah Caesarina Novi Marchianti",
"Danial H. Arsyi",
"Putu Bagus Dharma Permana",
"I Made Dwi Yudiartana Putra Susila",
"Octaviana Galuh Pratiwi",
"Diana Purwitasari",
"Nurina Hasanatuludhhiyah",
"Annette d'Arqom",
"Azlin Muhammad",
"Ancah Caesarina Novi Marchianti",
"Danial H. Arsyi",
"Putu Bagus Dharma Permana",
"I Made Dwi Yudiartana Putra Susila",
"Octaviana Galuh Pratiwi",
"Diana Purwitasari"
],
"abstract": "Introduction: Health literacy on the coronavirus disease 2019 (COVID-19) affects people’s capability to ascertain their health and health care quality during the pandemic. The objective of this study was to determine the levels of health literacy about COVID-19 vaccines and vaccinations (Vaccines and Vaccinations literacy-VL) in the Indonesian adult general population, assessing the perceptions of the respondents about current adult immunization and beliefs about vaccinations in general, and analyzing correlations of these variables with the VL levels.\nMethods: A cross-sectional study using a rapid survey was administered via the Internet. Data were analyzed using descriptive and inferential statistics; the internal consistency of the VL scales was evaluated using Cronbach’s alpha coefficient; the inter-correlation between the functional and interactive-critical VL questions, the underlying components (factors) and each question’s load on the components were identified using a Principal Component Analysis (PCA). An alpha level lesser than 0.05 was considered significant.\nResults: Responses to functional- and interactive/ critical- VL questions were acceptable and showed internal consistency (Cronbach’s alpha = 0.817 and 0.699, respectively), lowest values observed were 0.806 for functional scale and 0.640 for the interactive-critical scale. The PCA demonstrated that there were two components accounting for 52.45% of the total variability. Approximately 60% of respondents were females (n=686). Almost all respondents used the internet to seek information regarding COVID-19 and COVID-19 vaccinations. Many used at least one social media actively with 74.4% of respondents sometimes believing the validity of this information.\nConclusions: High scores were observed in both functional- and interactive/ critical-VL, and were quite in a balance between sexes in the prior VL and higher in females for the latter; these were also closely related to the educational level and age group. It is crucial to increase public health literacy in managing the pandemic.",
"keywords": [
"COVID-19",
"health risk",
"vaccines literacy",
"adult vaccinations",
"Indonesia"
],
"content": "Introduction\n\nSince the COVID-19 pandemic, the spread of vast information on this topic has increased, including vaccines and vaccinations.1,2 One must filter this information wisely to avoid fake news that might compromise the acceptance towards the COVID-19 vaccines and vaccinations.3 On the other hand, literacy levels on these subjects will also affect the opinion and personal beliefs when facing the issue.4 In the ongoing development of COVID-19 vaccinations and vaccines, evidence-based data released in a real-time fashion may lead to conflict when comprehended with no further authorized confirmation.5–7 These data are valuable to the decision-makers party to understand public sentiment and thus act accordingly to contain the pandemic.6 The current study aimed to determine and compare public opinion and sentiments on COVID-19, COVID-19 vaccines, and vaccinations before and after the national vaccinations program was held in Indonesia (in January 2021). These data represent the health literacy on the subjects implicating people’s skills and knowledge to gain and to use this information accordingly, which is critically valuable amidst a pandemic.7,8\n\nThe primary aim of this cross-sectional study was to elucidate the levels of public health literacy on COVID-19, COVID-19 vaccines and vaccinations in all regions of Indonesian (West, Central and East regions, respectively), which have yet to be analyzed largely based on specific age and sex groups. Here we assessed the respondents’ functional and critical/interactive-literacy, the individual perceptions and acceptance toward COVID-19 vaccines and vaccinations, individual perception and acceptance towards other vaccinations, individual’s belief towards health protocols and COVID-19 vaccinations safety and effectiveness, and the accessibility and usage of internet and social media to gain information related to COVID-19; to determine whether there have been gaps in the health literacy levels between groups and significant relations with the sociodemographic characteristics.\n\n\nMethods\n\nThis study was conducted in compliance with the Declaration of Helsinki (revised 2013 edition), the CHERRIES (Checklist for Reporting Results of Internet E-Survey), and STROBE (Strengthening the Reporting of Observational Studies in Epidemiology) guidelines.9–11 The principal objective of this cross-sectional study was to determine the levels of health literacy about COVID-19 vaccines and vaccinations (Vaccines and Vaccinations literacy-VL) in the Indonesian adult general population via a rapid survey distributed via the internet. Another objective was to assess the perceptions of the respondents about current adult immunization and beliefs about vaccinations in general, whilst analyzing their ability to seek and use information from the internet and social media; also to study any correlations of these variables with VL levels.\n\nWe conducted an anonymous online survey in which participants could select to take part or not. The questionnaire was built, administered, and collected via “SurveyPlanet Pro (Survey Planet, LLC)”, an electronic platform that creates web-based surveys that can be shared through other online services, including chatting applications, email messages, and web pages. A non-probability sampling method was adopted to spread the survey URL as a web-link collector that respondents could access and send their answers. The respondents were chosen by 10 coordinators of the survey based on the ownership of a valid email address (which was used to prevent multiple attempts to fill the survey by setting it accordingly in the Survey Planet), accessibility of the net, and age (older than 18 years old). They were Indonesian people with a good comprehension of Bahasa Indonesia who reside in five main islands in Indonesia that have been highly affected by COVID-19, including Java, Kalimantan, Sumatra, Sulawesi, and Papua regions. Respondents were free to complete the questionnaire and were asked to forward the link to others if they fulfilled the above inclusion criteria. No other exclusion criteria were adopted. Respondents were asked to provide honest answers and have been given informed consent and consent for information for both study participation and publication of the survey results prior to completing the survey. All respondents understood and agreed to the informed consent and consent for information of study participation and publication of the results indicated by clicking the “Yes” and “Next” button to proceed to the survey items. The survey could be accessed and answered via PC, tablet, or smartphone.\n\nThe items in the questionnaire were adapted and translated by three medical doctors, one of them is a native speaker, from a study by Biasio et al.12 All items had been pretested on the total of 233 respondents who passed the inclusion and exclusion criteria prior to actual study. The initial ace validity and reliability tests showed that all items were valid and reliable, with internal consistency after test-retest and inter-rater analysis (r=0.131; Cronbach’s alpha>0.60). The questionnaire was comprised of sections i.e., the respondents were provided with the rationale and scope of this study with the informed consent and consent for information of the study participation and publication of the study results for scientific purposes. By clicking the agreement button, they would go to the following seven sections composed of (1) nine questions on the sociodemographic data (age groups, sex, last formal education levels, occupation, monthly family income, geographical residence, financial difficulty during pandemic, history of positive confirmation of respondent and/or family members, comorbidity); (2) three questions to assess functional VL with a 3 point Likert scale (1 = often, 2 = sometimes, 3 = never) that measured respondents semantic and language comprehension (Extended data)13; (3) a 3 point Likert scale (1 = never, 2 = sometimes, 3 = often) of 6 questions to assess the cognitive of the respondents via the interactive-critical VL items (Extended data)13; (4) eight questions to assess individual perceptions and acceptance toward COVID-19 vaccines and vaccinations; (5) four questions to assess individual perception and acceptance towards other vaccinations i.e. influenza; (6) three questions to assess individuals’ belief towards health protocols and COVID-19 vaccinations safety and effectiveness; and (7) three questions to assess the accessibility and usage of net and social media to gain information related to COVID-19. The adapted two questions from a self-reported questionnaire for adulthood vaccinations prepared on the Ishikawa test for chronic non-communicable diseases that has been validated for content and construct were used to evaluate the VL levels. Three items of the questionnaire were directed to evaluate the functional VL, and six questions evaluated the interactive-critical VL according to Nutbeam’s definition.12,14 The full questionnaire can be found in the Extended data.13\n\nEthical approval for the current study was granted by the Health Research Ethics Committee (KEPK), Faculty of Medicine, Universitas Airlangga, Indonesia (no. 145/EC/KEPK/FKUA/2021). Participants gave their informed written consent for both study participation and publication of the survey results prior to completing the survey.\n\nThe score was calculated from the mean value of the answers to each scale (range 1 to 3), a higher value standing for a higher VL level. In the previous studies, these variables were treated as numerical, where comparable tools were adopted.12 The SPSS software version 17.0 was used for statistical analysis, by means of descriptive tables summarizing percentages, means, standard deviations (SD), confidence intervals (CI); also, medians and non-parametric tests, as data did not homogenous and follow a normal distribution (see the Results section). The relationship between the VL scales with other ordinal/numerical variables was analyzed using Spearman's correlation test; Chi-squared, Kruskal-Wallis, and Mann-Whitney tests were used for categorical and comparison the ratio/ordinal variables, respectively. The internal consistency of the VL scales was calculated via Cronbach’s alpha coefficient; the functional- and interactive/critical-VL items inter-relations and the analysis of factors/underlying components and each question's load on these factors were identified using the Principal Component Analysis (PCA). For each analysis, an alpha level=.05 was considered significant.12\n\n\nResults\n\nA total of 1,143 answers were collected during the 12 weeks, starting September 2, 2021, mainly via social media and email. Most of the participants (n=512; 44.79%) answered during the second week. From Table 1, it was shown that the Q12, Q14, Q15 (questions to identify functional VL) had a strong effect on principal component 2; whilst Q16-Q21 (questions to identify interactive' critical-VL) showed a moderate to strong effect on the principal component 1. Responses to functional- and interactive/critical-VL questions exhibited good/acceptable internal consistency (Cronbach’s alpha=0.817 and 0.699, respectively), lowest values observed were 0.806 for functional scale and 0.640 for the interactive-critical scale. PCA analysis demonstrated two components accounting for 52.45% of the total variability. A varimax rotation was applied to determine relationship between items and showed that all functional VL questions were affluent on one component, whereas all interactive-critical questions were sided on the other component. The two distinguished factors loaded on the questions in each component could be discerned as predicted, i.e., close relation was observed between the questions inside each of the functional scale and of the interactive-critical one (Figure 1, Table 1).\n\nThe functional vaccine literacy questions (Q12, Q14, Q15) and the interactive/critical COVID-19 vaccination literacy questions contain two components (Factor 1 and Factor 2); the value in bold corresponds to each variable with the factor with the greatest correlation.\n\nProjection of functional VL questions (Q12, Q14, Q15) and interactive-critical VL questions (Q16-Q21) on two components (Factor 1 and Factor 2). Variables that are close to each other are significantly positively correlated.\n\nApproximately 60% of respondents were females (n=686) and the rests were males (n=457). Most respondents were 18-30 years (57%, n=652), 36.2% were 31-50 years (n=414), 4.5% were 51-59 years (n=51) and only 2.3% were above 59 years (n=26). The education levels were mostly at the secondary stage (52.6%, n=601), 34.4% were at the primary level (n=393), 10.6% (n=121) were at the tertiary level, and the rests were either at the lesser degree or others. The university students were the predominant respondents (40.2%, n=460), followed by employees of the private sectors (23.9%, n=273), the civil servants (13.6%, n=155), entrepreneurs (6.6%, n=75) and others (15.7%, n=180). Respondents were mostly from Western-Indonesia (89%, n=1017), whilst only 8.6% (n=98) and 2.4% (n=28) were from the Central and Eastern-Indonesia. Most of respondents had a middle average of family income (34.7%, n=397 and 30.5%, n=349), 9.7 % (n=225) and 12.5% (n=143) had lower income, and 2.5% had a high income (n=29). About 57% (n=652) of all respondents had financial difficulty during the pandemic and 43% had no financial problem (n=491). There were 65.9% (n=753) respondents claimed that they or their household members had not been contracted COVID-19, whilst 34.1% (n=390) had been positively confirmed (Table 2).\n\nMost of respondents claimed that they had no comorbidities (81.7%, n=934), 9.9% (n=113) stated that they do not know if they had comorbidities; the rests stated they had one or more comorbidity i.e. respiratory-related diseases (3.4%, n=39), controlled-hypertension (3.1%, n=36), 1.6% stated to have uncontrolled-hypertension (n=18), controlled-T2DM (1.3%, n=15), uncontrolled-T2DM (0.7%, n=8), autoimmune diseases (3.4%, n=39), under-treatment-cancer (0.6%, n=7), untreated cancer (0.4%, n=5), neurological diseases (1%, n=12), 0.3% with mental illness (n=3), 0.3% (n=4) with congenital diseases and the rests claimed to have hypercholesterolemia, hyper-uremia, heart diseases, etc. (3.7%, n=43) (Table 3).\n\nThere were 63.8% of respondents often use internet to search for information on COVID-19 and/or COVID-19 vaccinations (n=729), 33.2% were sometimes (n=380) and 3% never used it (n=34). Approximately 62.4% of respondents have social media account and had been using it often to engage to these topics (n=713), 33.2% (n=379) only sometimes, whilst 4.5% (n=51) never. About 74.4% (n=850) respondents sometimes believed these information, 19.1% (n=218) often, and 6.6% (n=75) had never been (Table 4).\n\nThe mean score of functional-VL was 2.41±0.49 (median 2.33); whilst the interactive/critical-VL mean score was 2.38±0.43 (median 2.5), out of a maximum of 3 (Table 5). The functional-VL in males and females were 2.4±0.5 and 2.41±0.49 (no significant differences); and the interactive/critical-VL was lower in males than females that were 2.33± 0.46 and 2.4±0.41 (p<0.05, two-way Mann-Whitney).\n\n* P<0.05, Mann-Whitney.\n\nFor the question “Do you think that is a possibility to have safe and effective COVID-19 vaccines?” 848 answered “yes” (74.2%), 228 answered “don’t know” (19.9%) and 67 (5.9%) answered “no”; significant differences were observed related with the functional-VL (p=0.008), with the interactive/critical-VL (p<0.001), and with the sexes (p=0.04), but not significantly different between age groups (p=0.18).\n\nFor the question “Are you willing to get COVID-19 vaccinations?” 1092 answered yes (95.5%), 33 answered “don’t know” (2.9%) and 18 (1.6%) answered “no”; significant differences were observed only with the interactive/critical-VL (p<0.01).\n\nFor the question “Do you think the government can successfully reach the vaccinations target evenly in all provinces?” 770 answered “yes” (67.4%), 233 answered “don’t know” (20.4%) and 140 (12.2%) answered “no”; a significant difference was observed only with the interactive/critical-VL (p<0.01).\n\nFor the question of “Are you willing to pay for COVID-19 vaccinations?” 454 answered “yes” (39.7%), 143 answered “don’t know” (12.5%) and 546 (47.8%) answered “no”; significant differences were observed related with the functional-VL (p=0.008), with the interactive/critical-VL (p<0.001), and with the sexes (p=0.04), but not significantly different between age groups (p=0.18). Significant differences were observed in the critical/interactive-VL (p<0.01) and between the sexes (p=0.045).\n\nFor the question “Do you think school-age children must get COVID-19 vaccinations?” 970 answered “yes” (84.9%), 70 answered “don’t know” (6.1%) and 103 (9%) answered “no”; significant differences were observed related with the functional-VL (p=0.008), with the interactive/critical-VL (p<0.001), and with the sexes (p=0.04), but not significantly different between age groups (p=0.18). Significant differences were found between the interactive/critical-VL (p<0.01) and between the age groups (p<0.01).\n\nFor the question “Do you think certain brands of COVID-19 vaccines are safer and more effective compared to the other brands?” 514 answered “yes” (45%), 419 answered “don’t know” (36.7%) and 210 (18.4%) answered “no”; significant differences were observed related with the functional-VL (p<0.01), with the interactive/critical-VL (p<0.001), and with the sexes (p<0.01), but not significantly different between age groups (p=0.937).\n\nFor the question of “Do you have more assurance towards certain brands of COVID-19 vaccines based on the information you obtain from the internet?” 537 answered “yes” (47%), 240 answered “don’t know” (21%) and 366 (32%) answered “no”; significant differences were observed related with the functional-VL (p=0.008), with the interactive/critical-VL (p<0.001), and with the sexes (p=0.04), but not significantly different between age groups (p=0.34).\n\nFor the question of “Do you have more assurance towards certain brands of COVID-19 vaccines based on the information you obtain from the authority?” 747 answered “yes” (65.4%), 197 answered “don’t know” (17.2%) and 199 (17.4%) answered “no”; significant differences were observed related both with the functional-VL (p<0.01) and with the interactive/critical-VL (p<0.001) (Table 6).\n\nPerception and acceptance towards the COVID-19 vaccines and vaccinations were mostly positive, with affirmative responses between approximately 74.2% and 95.5%. However, most respondents had higher hesitancy to pay for the vaccines and vaccinations (47.8%) and 454 respondents were willing-to-pay (39.7%). The acceptance on the information regarding these topics was higher when coming from the authority than on the internet (65.4% vs. 47%). Acceptance toward COVID-19 vaccinations was quite high, which were significantly correlated with both functional- and interactive/critical-VL (r=0.112, p<0.001 and r=0.264, p<0.01); but not significantly related with either education level or the age group (r=-0.04, p=0.898 and r=-0.31, p=0.299, respectively).\n\nOnly a minority of respondents agreed completely (Likert score 3/no) with the statements: “I am not favourable to COVID-19 vaccines because they are unsafe and/or ineffective” (6.7%, n=76), “There is no need to get COVID-19 vaccinations because natural immunity already exists” (6%, n=69), whilst complete disagreement with statement: “Health protocols i.e. wearing a mask in public, physical distancing and washing hands are important things to do, in addition to COVID-19 vaccinations can help to lower the morbidity” was only 3% (n=35). On the other hand, most respondents were in complete disagreement with the first two statements (80.1%, n=914 and 84.7%, n=968, respectively) and mostly agreed with the last statement above (94.1%, n=1076). Answers with “don’t know” on all of these statements were 13.2% (n=151), 9.3% (n=106) and 2.8% (n=32), respectively.\n\nThere were significant correlations between each of these three statements response with the interactive/critical-VL (r= 0.264, p<0.01; r=0.192, p<0.01; r=0.135, p<0.01, respectively). Significant correlations were observed between each of the first two statements response with the functional-VL (r=0.112, p<0.01 and r=0.103, p<0.01) (Table 7).\n\n* Statement 1: I don't want to be vaccinated because the COVID-19 vaccine is not safe and/or ineffective.\n\n** Statement 2: I don't need to be vaccinated against COVID-19 because I already have the body's natural immunity.\n\n*** Statement 3: Health protocols such as wearing masks, washing hands and keeping a distance and avoiding crowds can reduce the transmission of COVID-19 in addition to COVID-19 vaccination.\n\nIn Table 6, approximately 67.2% (n=768) of participants self-reported that they had been vaccinated the previous years for at least one of these vaccinations: tetanus, pneumonia, diphtheria, pertussis, polio, measles, TBC, HPV, meningitis, hepatitis; significant differences were found in the interactive/critical-VL (p<0.01), between age groups (0.012) and sexes (p=0.028). Approximately 10.5% (n=120) respondents stated their intention to get one of these vaccinations again in the next season; significant differences were observed in the interactive/critical-VL (p<0.01), between age groups (p=0.03) and sexes (p<0.01).\n\nAbout 17.3% (n=198) respondents have been vaccinated for influenza; 17.9% (n=205) claimed they intend to receive another influenza and/or pneumonia vaccinations during the next season. Significant differences were found in the interactive/critical-VL (p<0.01) and between sexes (p<0.01).\n\nThe correlations between functional-VL with either education level or the age group were negative, with significance showed with the latter (r=-0.022, p=0.448 and r=-0.68, p=0.022). The interactive/critical-VL had significant correlations with both education level and the age group (r=0.109, p≤0.001 and r=-0.062, p=0.037). Significant correlations were found between the functional-VL and the interactive/critical-VL (r=0. 084, p=0.005) (Figures 2-3).\n\n\nDiscussion\n\nCoronavirus disease 2019 (COVID-19) has become a worldwide challenge that has affected the health systems of many countries, including Indonesia.15,17 SARS-CoV-2 mutated virus produces variants with dynamic responses to available vaccines.18 Although research is still emerging, the reported data show that people with chronic and comorbid illnesses are highly susceptible to COVID-19,19,20 and experience higher levels of disease severity when they get infected.21–23 Antiviral treatments and vaccines that are proven to be fully effective against viral mutations are under ongoing development.24,25 Various scientific studies continue to grow every day; however, the research results are still mixed and often raise questions in the community.26,27 The need to master the skill to understand, access, and act accordingly based on valid health information (health literacy) is emerging to be a vital psychosocial factor of health outcomes, determined by sociodemographic characteristics i.e., age, ethnicity, and economic levels.7,28 The proposed effective way to contain this problem is to build good health literacy about COVID-19 and COVID-19 vaccinations, including via this online survey, as the public can contribute to the preventive and promotive measurement by protecting themselves while protecting others, to halt the spread of COVID-19.27,29 As a result, health literacy is crucial because it becomes the centre value in filtering information about COVID-19 and COVID-19 vaccinations. Access to this information also plays a vital role in the level of health literacy,30–32 although hoaxes can produce misunderstandings.33,34\n\nAs an archipelago, Indonesia has a geographical disparity with cultural diversity that influences values and behaviour in facing external challenges i.e., COVID-19.15,35 In addition, socioeconomic and educational levels determine literacy.36 Individual compliance to health protocols, including physical distancing, voluntary screening tests, self-isolation if infected, face mask use in public places, and hand sanitation are various.16,37 Together with the national vaccinations program, the infection rate has decreased by more than 90% since July 2021 when the Delta variant began to strike.38 Restriction measures are applied by requiring returning travellers to do a quarantine, restricting public recreational places, non-essential sectors, offices, and schools to implement emergency micro-based social activity restrictions (PPKM) since 2020.39 However, the disparity of engagement levels in these efforts might produce hotspots with additional waves of mutated-virus infection, certain clusters might be suffering more severely affected by COVID-19.40,41\n\nThis rapid-on-line survey was responded predominantly by female participants and the age group between 18-50 years. They mostly graduated from the primary and secondary education levels, and this was presented in the predominant occupation of the respondents were university students, while private and public employees came next. The lack of direct promotion and distribution of the online survey during the pandemic might be the reason of the predominant respondents were from Java, Sumatra, and Kalimantan than from other regions i.e. Sulawesi, Maluku, Bali, Nusa Tenggara, and Papua.42 However, this could serve the purpose of this study to capture the data from where the highest incidence of COVID-19 was reported from that was in Western Indonesia. The monthly family income was mostly at between 2-24 million (IDR), which is classified as a middle-class economy, this could be affected by the pandemic as stated by more than half of respondents who also claimed that this pandemic had struck at least one of their household family members.\n\nApproximately more than 80% of all respondents stated that they were free from any comorbidity i.e., respiratory-related diseases (asthma, COPD), hypertension, cancer, T2DM, psycho-neurology disorders, congenital abnormality, metabolic syndrome, and other pathological conditions, although about 10% was aware of these comorbidities. During the pandemic, an increasing number of people relied on the internet and social media to obtain information about COVID-19. In our study, we found that all respondents used the internet to seek information regarding COVID-19 and COVID-19 vaccinations, amongst these, they used at least one social media actively, with a high percentage of respondents sometimes believing the validity of this information (74.4%).43,44\n\nFrom the maximum number of the Likert scale of the vaccines literacy (VL), we observed high scores in both functional- and interactive/critical-VL where these were quite in a balance between sexes in the prior VL and higher in females for the latter. Perception and acceptance toward COVID-19 vaccines and vaccinations were generally positive shown from the predominant good responses on the safety and effectiveness of the vaccines and from the willingness to be vaccinated. However, when asked to pay for the vaccinations, almost half of the respondents disagreed (47.8%), 20.4% were still in doubt, whilst the other 39.7% agreed. More than 80% of respondents agreed that school-aged children must be vaccinated against COVID-19; 45% agreed that certain vaccine brands have more safety and effectiveness than the others (mostly due to the RNA-based vs. weakened- and/or killed virus-based vaccines) whilst about 47% respondents felt more self-assurance after they read the information on certain vaccines via internet. The acceptance on the information regarding these topics was higher when coming from the authority than the internet (65.4% vs. 47%); acceptance toward COVID-19 vaccinations was significantly correlated with both functional- and interactive/critical-VL but not significantly related to either education level or the age group. These data showed us that even though lots of people seek information using the internet, whilst might decrease their anxiety about the unknown part of COVID-19 vaccines and vaccinations, the validity of the information is questionable and they preferred the information from the authority.45,46 Here, perception and acceptance of COVID-19 vaccines and vaccinations of Indonesian people were shown to have more strong correlation with the interactive/critical-VL rather than the functional-VL or other socio-demography characteristics i.e., sex and age group.38\n\nThe disagreement of getting COVID-19 vaccinations due to its safety and/or effectiveness also due to the belief in natural immunity was quite low, whilst most respondents agreed at the value of conducting health protocols regardless of the vaccination states. The first two statements' perception and belief were significantly correlated with the functional VL and the interactive/critical VL, while the last statement was only significantly correlated with the functional VL. Arguably, the values play a vital role in facing the pandemic and speeding up the national vaccinations coverage that has started since the beginning of 2021 in Indonesia.47,48 More respondents had been vaccinated against various infectious diseases, although only about 17.3% had been vaccinated for the influenza. Only about 10-18% of respondents claimed their intention to have these vaccinations again in the next season; these were significantly correlated with the interactive/ critical-VL and sex. These data might show us that most healthy persons had relatively low literacy on this topic; these vaccinations have more frequently done amongst school-age children than adults in Indonesia. Previous study on the consumption of supplements during the COVID-19 pandemic in Indonesia found that these behaviours was influenced by education, age, family income, and family expense.49 We observed that both VL were significantly correlated with the age group, the highest was found in the 18-50 years age groups; whilst the interactive/critical-VL had a parallel and significant correlation with the education level.\n\nTo the best of our knowledge, this study is the first to analyze health literacy on COVID-19 and COVID-19 vaccines and vaccinations. The current study was carried out in a period between the first and the second dose of the COVID-19 vaccinations program was running. The results would be valuable for implementing boosters involving large populations where further studies are called. The VL has been used to show health literacy on various studies. Here it represented people's acceptance, perception and attitude towards COVID-19 and its vaccinations. The hesitance to the vaccinations was shown due to i.e., personal belief and knowledge, willingness-to-pay, accessibility, and the influence from the community and the authority. A vast amount of information on COVID-19 potentially confused people to the extent of believing or rejecting all news related to the disease.50 Health literacy can assist people in filtering and finding correct information and then using these accordingly; including logically reason the vaccinations and health protocols.12,51,52 Health education must be adjusted according to local wisdom and condition to meet the needs of diverse clusters hence could bridge the gap in the community with various health literacy.6,50\n\nThis study adapted the convenience sampling in which university students were the predominant respondents. This was due to the practical reasons that these respondents are the most likely ones who regularly use the internet and social media and were familiar with the online survey. Another limitation is that the respondent’s demography was mostly from Java and least from the eastern part of Indonesia. Self-claimed responses might affect the generalization of study results. However, the PCA of both VL scales showed good internal consistency and component loading.\n\nFurthermore, findings from the current study are valuable due to its topics and the relatively broad range of respondents in terms of education levels and family income can represent most part of the population; levels of VL and their associations to various independents variables were comparable to other similar studies, validated using direct interview methods. Uneven levels of health literacy are an adverse situation that might slow the successful rate against the pandemic. Further studies are needed especially on the comprehension of the importance of vaccinations and health protocols that can help people to adapt to the new normal era.\n\n\nConclusions\n\nA self-stated online survey conducted in the current study can provide reliable data on the level of health literacy of quite a large sample population distributed in different islands. The findings of this study could help community and decision-maker parties prepare communication and action strategies to cope with the pandemic. The VL levels of all respondents showed strong relationships with the individual perception, belief, and acceptance toward vaccinations and health protocols. Valid information regarding COVID-19 from authority are called for and could help the public to act accordingly.",
"appendix": "Data availability\n\nDryad: Survey Data of the Health Literacy on COVID-19 and COVID-19 Vaccination in Indonesia, https://doi.org/10.5061/dryad.2fqz612sg. 13\n\nThis project contains the following underlying data:\n\n- Full response data.xlsx\n\nDryad: Survey Data of the Health Literacy on COVID-19 and COVID-19 Vaccination in Indonesia, https://doi.org/10.5061/dryad.2fqz612sg. 13\n\nThis project contains the following extended data:\n\n- English translation of the full questionnaire.pdf\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 would like to thank to Moh. Reza Farabi, Anggit Satrio Yudhono and Natasha Nurvita Brilianti for their valuable contributions in disseminating the questionnaires. We have obtained permission from Moh. Reza Farabi, Anggit Satrio Yudhono and Natasha Nurvita Brilianti to be acknowledged here. Thank you to LPPM, Universitas Airlangga for the valuable support.\n\n\nReferences\n\nRathore FA, Farooq F: Information overload and infodemic in the COVID-19 pandemic. J. Pak. Med. Assoc. 2020 May; 70(Suppl 3(5)): 1–5. Publisher Full Text\n\nWidiawaty MA, Lam KC, Dede M, et al.: Spatial differentiation and determinants of COVID-19 in Indonesia. BMC Public Health. 2022; 22(1): 1016–1030. PubMed Abstract | Publisher Full Text\n\nMontagni I, Ouazzani-Touhami K, Mebarki A, et al.: Acceptance of a COVID-19 vaccine is associated with ability to detect fake news and health literacy. J. Public Health (Oxf.). 2021 Dec; 43(4): 695–702. Publisher Full Text\n\nFojnica A, Osmanovic A, Đuzic N, et al.: COVID-19 vaccine acceptance and rejection in an adult population in Bosnia and Herzegovina. PLoS One. 2022 Feb 28; 17(2): e0264754. PubMed Abstract | Publisher Full Text\n\nHuang Q, Lei S, Ni B: Perceived information overload and unverified information sharing on WeChat amid the COVID-19 pandemic: A moderated mediation model of anxiety and perceived herd. Front. Psychol. 2022; 13. PubMed Abstract | Publisher Full Text\n\nHarisanty D, Srirahayu DP, Anna NEV, et al.: Digital literacy for COVID-19 information in Indonesian society. DigitalCommons@University of Nebraska - Lincoln;2021.Reference Source\n\nAlqudeimat Y, Alenezi D, Alhajri B, et al.: Acceptance of a COVID-19 vaccine and its related determinants among the general adult population in Kuwait. Med. Princ. Pract. 2021; 30(3): 262–271. PubMed Abstract | Publisher 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 Jul 14; 8: 381. PubMed Abstract | Publisher Full Text\n\nEysenbach G: Improving the quality of web surveys: the Checklist for Reporting Results of Internet E-Surveys (CHERRIES). J. Med. Internet Res. 2004; 6: e34. PubMed Abstract | Publisher Full Text\n\nWorld Medical Association: World Medical Association Declaration of Helsinki: Ethical principles for medical research involving human subjects. JAMA. 2013 Nov; 310(20): 2191–2194.\n\nVandenbroucke JP, von Elm E , Altman DG, et al.: Strengthening the Reporting of Observational Studies in Epidemiology (STROBE): Explanation and Elaboration. PLoS Med. 2007 Oct 16; 4(10): e297. PubMed Abstract | Publisher Full Text\n\nBiasio LR, Bonaccorsi G, Lorini C, et al.: Assessing COVID-19 vaccine literacy: a preliminary online survey. Hum. Vaccin. Immunother. 2021; 17(5): 1304–1312. PubMed Abstract | Publisher Full Text\n\nKalanjati VP, Hasanatuludhhiyah N, D’Arqom A, et al.: Survey Data of the Health Literacy on COVID-19 and COVID-19 Vaccination in Indonesia.2022.dryad.orgReference SourcePublisher Full Text\n\nIshikawa H, Takeuchi T, Yano E: Measuring functional, communicative, and critical health literacy among diabetic patients. Diabetes Care. 2008 May; 31(5): 874–879. PubMed Abstract | Publisher Full Text\n\nDjalante R, Lassa J, Setiamarga D, et al.: Review and analysis of current responses to COVID-19 in Indonesia: Period of January to March 2020. Prog. Disaster Sci. 2020; 6: 100091.Reference Source\n\nElgaputra RR, Adhi Sakti EY, Widyandri DB, et al.: Implementasi sosialisasi COVID-19 dalam upaya meningkatkan kesadaran masyarakat terhadap protokol kesehatan di Kota Jakarta. J. Layanan Masy (Journal Public Serv.). 2020 Nov 29; 4(2 SE-Articles): 423–433. Publisher Full Text Reference Source\n\nHarvey WT, Carabelli AM, Jackson B, et al.: SARS-CoV-2 variants, spike mutations and immune escape. Nat. Rev. Microbiol. 2021; 19(7): 409–424. PubMed Abstract | Publisher Full Text\n\nMoore JP, Offit PA: SARS-CoV-2 vaccines and the growing threat of viral variants. JAMA. 2021 Mar 2; 325(9): 821–822. PubMed Abstract | Publisher Full Text\n\nJasti M, Nalleballe K, Dandu V, et al.: A review of pathophysiology and neuropsychiatric manifestations of COVID-19. J. Neurol. 2021; 268(6): 2007–2012. PubMed Abstract | Publisher Full Text\n\nMukaetova-Ladinska EB, Kronenberg G: Psychological and neuropsychiatric implications of COVID-19. Eur. Arch. Psychiatry Clin. Neurosci. 2021; 271(2): 235–248. PubMed Abstract | Publisher Full Text\n\nAlonso-Lana S, Marquié M, Ruiz A, et al.: Cognitive and neuropsychiatric manifestations of COVID-19 and effects on elderly individuals with dementia. Front. Aging Neurosci. 2020; 12(October). Publisher Full Text\n\nIndarwati R: Lindungi lansia dari Covid-19. Indones. J. Community Heal. Nurs. 2020 Feb 1; 5(1 SE-Editorial).Reference Source\n\nSensusiati AD, Amin M, Nasronudin N, et al.: Age, neutrophil lymphocyte ratio, and radiographic assessment of the quantity of lung edema (RALE) score to predict in-hospital mortality in COVID-19 patients: a retrospective study. F1000Res. 2020; 9: 1286. Publisher Full Text\n\nHussman JP: Cellular and molecular pathways of COVID-19 and potential points of therapeutic intervention. Front. Pharmacol. 2020; 11(July): 1–17.\n\nAndrei G: Vaccines and antivirals: Grand challenges and great opportunities. Front. Virol. 2021; 1. Publisher Full Text\n\nSaba L, Gerosa C, Fanni D, et al.: Molecular pathways triggered by COVID-19 in different organs: ACE2 receptor-expressing cells under attack? A review. Eur. Rev. Med. Pharmacol. Sci. 2020; 24(23): 12609–12622. PubMed Abstract | Publisher Full Text\n\nSolís Arce JS, Warren SS, Meriggi NF, et al.: COVID-19 vaccine acceptance and hesitancy in low- and middle-income countries. Nat. Med. 2021; 27(8): 1385–1394. PubMed Abstract | Publisher Full Text\n\nYağar F: Fear of COVID-19 and its association with health literacy in elderly patients. J. Patient Exp. 2021 Jan 1; 8: 23743735211056504. PubMed Abstract | Publisher Full Text\n\nSondakh JJS, Warastuti W, Susatia B, et al.: Indonesia medical students’ knowledge, attitudes, and practices toward COVID-19. Heliyon. 2022; 8(1): e08686. PubMed Abstract | Publisher Full Text\n\nSpink J, Cloney D, Berry A: Beyond letters and numbers: the COVID-19 pandemic and foundational literacy and numeracy in Indonesia.2022.\n\nWijaya MC, Kloping YP: Validity and reliability testing of the Indonesian version of the eHealth Literacy Scale during the COVID-19 pandemic. Health Informatics J. 2021; 27(1): 1460458220975466. PubMed Abstract | Publisher Full Text\n\nFukuda Y, Ando S, Fukuda K: Knowledge and preventive actions toward COVID-19, vaccination intent, and health literacy among educators in Japan: An online survey. PLoS One. 2021 Sep 20; 16(9): e0257552. PubMed Abstract | Publisher Full Text\n\nDewayani A, Ferdinandus ED, Prastio RP, et al.: Maximizing millennial students role in combating COVID-19 hoaxes and myths. Biomol. Heal. Sci. J. 2021; 4(1): 42. Publisher Full Text\n\nPagoto S, Waring ME, Xu R: A call for a public health agenda for social media research. J. Med. Internet Res. 2019; 21(12): e16661. PubMed Abstract | Publisher Full Text Reference Source\n\nRahmawati D, Mulyana D, Lumakto G, et al.: Mapping Disinformation During the Covid-19 in Indonesia: Qualitative Content Analysis. J ASPIKOM. 2021 Jul 25 [cited 2022 Jan 26]; 6(2): 222–234. Publisher Full Text Reference Source\n\nMohamed NA, Solehan HM, Mohd Rani MD, et al.: Knowledge, acceptance and perception on COVID-19 vaccine among Malaysians: A web-based survey. PLoS One. 2021 Aug 13; 16(8): e0256110. PubMed Abstract | Publisher Full Text\n\nSeale H, Heywood AE, Leask J, et al.: COVID-19 is rapidly changing: Examining public perceptions and behaviors in response to this evolving pandemic. PLoS One. 2020 Jun 23; 15(6): e0235112. PubMed Abstract | Publisher Full Text\n\nIndonesian Ministry of Health: COVID-19 vaccine acceptance survey in Indonesia. Kementeri Kesehat Republik Indones. 2020; 1(1): 1–8.Reference Source\n\nMiharja M, Salim E, Nachrawi G, et al.: Implementation of emergency public activity restrictions (PPKM) in accordance with human rights and Pancasila principles. BIRCI-Journal. 2021; (15): 6855–6866. Publisher Full Text\n\nRembulan G, Wijaya T, Palullungan D, et al.: Kebijakan pemerintah mengenai Coronavirus Disease (COVID-19) di setiap provinsi di Indonesia berdasarkan analisis klaster. JIEMS (Journal Ind. Eng. Manag. Syst.). 2020 Sep 7; 13. Publisher Full Text\n\nSevindik I, Tosun MS, Yilmaz S: Local response to the covid-19 pandemic: The case of Indonesia. Sustain. 2021; 13(10). Publisher Full Text\n\nUNICEF-UNDP-Australia Indonesia Partnership for Economic Development-The SMERU Research Institute:2021; Socioeconomic impact of the COVID-19 pandemic on households in Indonesia: Three rounds of monitoring surveys. Unicef.Reference Source\n\nNeely S, Eldredge C, Sanders R: Health information seeking behaviors on social media during the COVID-19 pandemic among American social networking site users: Survey study. J. Med. Internet Res. 2021 Jun; 23(6): e29802. PubMed Abstract | Publisher Full Text\n\nBright HR, Chandy SJ, Pradeep R, et al.: Online health information seeking behaviour due to COVID-19 pandemic-induced health related anxiety among the general population in India. J. Assoc. Physicians India. 2022 Jan; 70(1): 11–12.\n\nLindholt MF, Jørgensen F, Bor A, et al.: Public acceptance of COVID-19 vaccines: Cross-national evidence on levels and individual-level predictors using observational data. BMJ Open. 2021 Jun 1; 11(6): e048172. PubMed Abstract | Publisher Full Text Reference Source\n\nSidarta C, Kurniawan A, Lugito NPH, et al.: The determinants of COVID-19 vaccine acceptance in Sumatra. Kesmas. 2022; 17(1): 32–39. Publisher Full Text\n\nAkther T, Nur T: A model of factors influencing COVID-19 vaccine acceptance: A synthesis of the theory of reasoned action, conspiracy theory belief, awareness, perceived usefulness, and perceived ease of use. PLoS One. 2022 Jan 12; 17(1): e0261869. PubMed Abstract | Publisher Full Text\n\nSiewchaisakul P, Sarakarn P, Nanthanangkul S, et al.: Role of literacy, fear and hesitancy on acceptance of COVID-19 vaccine among village health volunteers in Thailand. PLoS One. 2022 Jun 24; 17(6): e0270023. PubMed Abstract | Publisher Full Text\n\nD’Arqom A, Sawitri B, Nasution Z, et al.: “Anti-COVID-19” medications, supplements, and mental health status in Indonesian mothers with school-age children. Int. J. Women's Health. 2021; 13(June): 699–709. PubMed Abstract | Publisher Full Text\n\nvan der Linden S , Roozenbeek J, Compton J: Inoculating against fake news about COVID-19. Front. Psychol. 2020; 11(October): 1–7. Publisher Full Text\n\nIfroh RH, Asrianti T: Health literacy, media exposure and behavior among young adults during the COVID-19 pandemic. J. Ilmu Kesehat. Masy. 2020; 11(3): 223–236. Publisher Full Text\n\nKhaerunnisa S, Syafa’ah I, Wungu CDK, et al.: The improvement of community knowledge, attitudes and practices after COVID-19 socialization. Folia Medica Indones. 2021 Jun 1; 57(2 SE-Original Research): 95–103. Publisher Full Text Reference Source"
}
|
[
{
"id": "155593",
"date": "01 Dec 2022",
"name": "Athira Nandakumar",
"expertise": [
"Reviewer Expertise Epidemiology and Preventive 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\nINTRODUCTION\nThe importance of public health literacy especially during a pandemic is the most relevant and the authors through this online survey study very well showed how it prevailed in Indonesian islands together with COVID-19.\nComment: The introduction part lacks the background or already existing health literacy information in Indonesia.\nMETHODS\n1) Data Collection:\nComment: availability of net should be changed to internet.\n\nQuestion: The study seems to be done in the major areas like Java, Kalimantan, Sumatra, Sulawesi, and Papua regions, so the participants' number might have been less, N is just 1143. What was the response rate?\n\nQuestion: What were the selection criteria for the respondents, and how they conformed their residence?\n2) Data Analysis:\nQuestion: How the authors treated the effect of confounding factors is not clear.\n\nComment: Probably regression analysis adjusting for potential confounding variables can be an alternative model for analysis.\n3) Results:\nTable 2, Question: The residence area names mentioned in Table – western, eastern and central Indonesia doesn’t correspond to the 5 area names mentioned in the Introduction. Please use a uniform style in names. The table shows the main area is the western region with around 90 percent participants\n\nTable 5, Comment: Adding some simple definitions of Functional, Interactive, or critical vaccine literacy scores will be easier for the readers.\n\nTable 5, Question: Just the Median of the vaccine literacy scores makes it clear, and hence mean is not necessary. Or is it based on some analysis?\n\nTable 5, Question: Functional vaccine literacy score for the total population is 2.41. Is that a good score? Is it comparable?\n4) Perception and acceptance towards COVID-19 vaccines and vaccinations:\nComment: Citing Table 6 alone is sufficient to portray the purpose.\n5) Correlation of VL with other variables:\nFigure 2, Question: Why age groups are selected; especially because 57% are in the first group, education and occupational status could have been better alternatives.\n\nFigure 2,3 Comment: Y-axis labels can be just 0,1,2,3;\n\nFigure 2,3 Question: Why the authors choose the order of categories in the X-axis is not clear.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes",
"responses": [
{
"c_id": "9144",
"date": "20 Dec 2022",
"name": "Viskasari Kalanjati",
"role": "Author Response",
"response": "Athira Nandakumar, Department of Epidemiology and Preventive Medicine, Kagoshima University Graduate School of Medical and Dental Sciences, Kagoshima, Japan INTRODUCTION The importance of public health literacy especially during a pandemic is the most relevant and the authors through this online survey study very well showed how it prevailed in Indonesian islands together with COVID-19. Comment: The introduction part lacks the background or already existing health literacy information in Indonesia. Author comment: Thank you, we have made a revision METHODS 1) Data Collection: Comment: availability of net should be changed to internet. Author’s response: Thank you, already revised Question: The study seems to be done in the major areas like Java, Kalimantan, Sumatra, Sulawesi, and Papua regions, so the participants' number might have been less, N is just 1143. What was the response rate? Author’s response: The response rate was 1143/1153 (99%). A total of 1153 respondents gave responses, however, 10 of them declined to finish the survey due to technical matters, hence the number of total respondents included in our analysis was 1143. Question: What were the selection criteria for the respondents, and how they conformed their residence? Author’s response: Thank you for your question, the answers of these have been detailed in the methods, as followed: ’… The respondents were chosen by 10 coordinators of the survey based on the ownership of a valid email address (which was used to prevent multiple attempts to fill the survey by setting it accordingly in the Survey Planet), accessibility to the internet, and age (older than 18 years old). They were Indonesian citizens with a good comprehension of Bahasa Indonesia who reside in the western (Sumatera, Java, and Kalimantan islands), central (Bali, West Nusa Tenggara, Sulawesi Islands), and eastern (East Nusa Tenggara, Maluku, Papua Islands) regions of Indonesia”. We asked all respondents to give honest answers whilst 10 in-charged surveyors confirmed the residence of each respondent personally as we did not require them to submit any personal identity card as proof due to security reasons. 2) Data Analysis: Question: How the authors treated the effect of confounding factors is not clear. Comment: Probably regression analysis adjusting for potential confounding variables can be an alternative model for analysis Author’s response: Thank you for your comment. We have applied homogeneity and normality tests to control the variance and distribution of the data set whilst filtering the respondent’s background variability using strict inclusion and exclusion criteria, thus minimizing potential bias. We then applied non-parametric inferential statistics to compare the median of the variables and employed a level of significance of p < 0.05 with 95% of CI. 3) Results: Table 2, Question: The residence area names mentioned in Table – western, eastern and central Indonesia doesn’t correspond to the 5 area names mentioned in the Introduction. Please use a uniform style in names. The table shows the main area is the western region with around 90 percent participants Author’s response: Thank you, we have made a revision accordingly. Table 5, Comment: Adding some simple definitions of Functional, Interactive, or critical vaccine literacy scores will be easier for the readers. Author’s response: Thank you for your comment. We have detailed the definition Functional, Interactive, or critical vaccine literacy scores in the method section in the paragraph right before the ethical consideration. Table 5, Question: Just the Median of the vaccine literacy scores makes it clear, and hence mean is not necessary. Or is it based on some analysis? Author’s response: Thank you for your comment. The comparison of means was done in the parametric data set, while the medians were compared in non-parametric data set, thus implied in Table 5. Table 5, Question: Functional vaccine literacy score for the total population is 2.41. Is that a good score? Is it comparable? Author’s response: Thank you for your question. The Functional vaccine literacy score were assessed using three questions with a 3-point Likert scale (1 = often, 2 = sometimes, 3 = never) that measured respondents’ semantic and language comprehension; thus, 2.41 score of the total population represented that the major respondents never or only sometimes found difficulties regarding the semantic and the language when understanding COVID-19 vaccines and vaccination information. 4) Perception and acceptance towards COVID-19 vaccines and vaccinations: Comment: Citing Table 6 alone is sufficient to portray the purpose. Author’s response: We agree, thank you very much. 5) Correlation of VL with other variables: Figure 2, Question: Why age groups are selected; especially because 57% are in the first group, education and occupational status could have been better alternatives. Author’s response: Thank you for your comment. We have chosen analysis based on the age groups for this particular figure, however, we also detailed further analysis based on the education and monthly income comparison in Table 7. Figure 2,3 Comment: Y-axis labels can be just 0,1,2,3; Author’s response: Thank you for your comment, we have revised accordingly. Figure 2,3 Question: Why the authors choose the order of categories in the X-axis is not clear. Author’s response: Thank you for your comment, we have revised accordingly."
}
]
},
{
"id": "155592",
"date": "06 Dec 2022",
"name": "Puspa Sari",
"expertise": [
"Reviewer Expertise 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\nIn this study the Authors investigated Health literacy on COVID-19 and COVID-19 vaccinations in Indonesia. This is an interesting and relevant article according to the current condition of the covid pandemic, and this will support the efforts of health workers to increase public knowledge about covid 19 and covid 19 vaccinations. However, I have the following comments and questions of the manuscript:\nAbstract: The title and abstract cover the main aspect of the work.\n\nIntroduction: Background and study-related information are provided in the introduction. But the author may clarify whether earlier research similar to this has been done. If not, explain that this is a novelty of this research. Explain whether there are any official information sources in Indonesia that the general public can access.\n\nMethods: Please explain the sample size. Is there no minimal sample?\nResults:\nProvides new, helpful facts for Indonesia\nDiscussion: Relevant discussion. Limitations and implications are described.\n\nIs the work 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": "9143",
"date": "20 Dec 2022",
"name": "Viskasari Kalanjati",
"role": "Author Response",
"response": "Puspa Sari, School of Medicine and Public Health, University of Newcastle, Callaghan, Australia Introduction: Background and study-related information are provided in the introduction. But the author may clarify whether earlier research similar to this has been done. If not, explain that this is a novelty of this research. Explain whether there are any official information sources in Indonesia that the general public can access. Author’s response: Thank you for the insightful advice, we have added a previous study from Indonesian sample population that would explain about this in the inteoduxtion, as followed: \" In the study by Harisanty et al., (2021), it was reported that the digital literacy level of COVID-19 amongst 500 respondents in east Java, Indonesia was 3.69 and represented a good average level. 5 – 7 \" Methods: Please explain the sample size. Is there no minimal sample? Author’s response: Thank you, we have added it to the method section, as followed: \"...The minimum sample size was calculated using a sample size calculator (http://www.raosoft.com/samplesize.html) with 3% margin of error and 95% confidence level. The minimum sample size required in this study was 1014.\""
}
]
}
] | 1
|
https://f1000research.com/articles/11-1296
|
https://f1000research.com/articles/11-1577/v1
|
23 Dec 22
|
{
"type": "Research Article",
"title": "Detection and identification of cyclops in well’s water, Jabal Awliya locality, Khartoum, Sudan 2021",
"authors": [
"Athar Salah Eldin Moustafa",
"Saada Mohamed Nour",
"Gihan Mahmoud Idris",
"Babiker Mohamed Hussein",
"Muaiad Karar Ahmed Mohamed",
"Salma Mohamed Ahmed Mohamedelrasheed",
"Athar Salah Eldin Moustafa",
"Saada Mohamed Nour",
"Gihan Mahmoud Idris",
"Babiker Mohamed Hussein"
],
"abstract": "Background: Dracunculus medinensis or \"Guinea-worm\" is the parasitic worm that causes Dracunculiasis disease. The Cyclops (Meso and Micro Cyclops), which is only spread through drinking from water sources in endemic areas, carries this worm. This study aimed to detect and identify Cyclops in wells water, Jabal Awliya locality Khartoum state, Sudan 2021 Methods: This is a cross sectional study held in Jabal Awliya locality which is located in Khartoum state Sudan from 2021-2022. The locality is one of the seven localities in the state which is considered as the main gate for the refugees from South Sudan, Chad and Ethiopia where Dracunculus medinensis has not been eradicated yet. The population is 274,321. Results: A total of 264 samples were collected,132 from well’s water and 132 from post wells water sources. Well’s water contamination with cyclops was found to be 2.4% while post well’s contamination was found to be 8.9%. The species reported by microscopy are: Afrocyclops, Mesocyclops and Microcyclops. Significant statistical difference between well’s and post well’s contamination was found (p-value 0.02). Conclusions: In conclusion, it is apparent that well’s water as well as dam water are contaminated with copepods (Afro-, Meso- and Microcyclops). Hence Residents in the research regions are at hazard for Dracunculus infection whenever larvae make their way to the water sources since mesocyclops and microcyclops are recognized Guinea worm vectors. Since no apparent water treatment system has been found in the areas under study, it is crucial that water sources in this area be treated before consumption in order to reduce the risk of illness. Awareness by the worm as well as the disease should be raised in order to ensure that the larvae from the hosts will not be carried to the wells.",
"keywords": [
"Cyclops",
"dracunculus Medinensis",
"wells",
"Sudan",
"copepods",
"mesocyclops",
"microcyclops"
],
"content": "Introduction\n\nDracunculus medinensis or “Guinea-worm” is a parasitic worm that causes Dracunculiasis disease. It is one of the large tissue parasites that affect human beings. The adult female is between 600 and 800 mm long and 2 mm in diameter. There are around 3 million embryos inside. The cycle is completed by the parasite migrating through the victim's subcutaneous tissues after ingestion of the infected stage, which causes excruciating pain, especially in the joints. Most of the time, the worm eventually pops out of the feet. It results in fever, nausea, and vomiting along with an extremely painful oedema, blister, and ulcer.\n\nDracunculus and dracunculiasis issue has been discussed in the seminal review by Ralph Muller via serial publication since 1971. In order to promote a campaign to eradicate dracunculiasis, the Centers for Disease Control and Prevention and the Carter Center formed a partnership of organizations. In 1986, endemic dracunculiasis was reported to exist in 18 of 20 nations, including Sudan (Ruiz-Tiben and Hopkins 2006). Elimination of 98% of the estimated 3.5 million cases of dracunculiasis (Guinea worm disease) that existed less than 20 years ago has been achieved. The majority of the remaining patients are in southern Sudan, and the global eradication campaign cannot be finished until the Sudanese civil war is over, despite the fact that seven of the 20 countries that were endemic for the disease have already eradicated the disease (Hopkins and Withers 2002).\n\nThe primary source of water supply in Africa is still surface water, which is obtained through small-scale dams, hafirs, ponds, and wells, many of which are man-made. Given that they are dispersed widely and primarily affect isolated rural communities, their impact on human health has gone unnoticed. According to World Health Organization studies, the disaster is that water can spread more than 80 dangerous diseases. Dracunculiasis is one of the illnesses and is brought on by the worm Dracunculus medinensis. The Cyclops (Meso and Micro Cyclops), which are only transmitted through drinking from water sources in endemic areas, carries this worm. It is well recognized that small-scale dams and lagoons contribute to the prevalence of dracunculiasis (Ilegbodu et al. 1987, Cairncross and Tayeh 1988). Theoretically, tiny dams put a lot more people at risk of disease transmission than huge dams, however this has been contested (Jewsbury and Imevbore 1988). On the other hand, not many studies have discussed the transmission of the disease through the wells.\n\nAs the result of southern Sudan war many of south Sudanese travel to north Sudan, especially the Jabal Awliya locality, that became a refugee camp during the Second Sudanese Civil War, housing more than 100,000 inhabitants. It became a gate way for South Sudanese refugees. The presence of a source of infection -which is the refugees in the camps or another hosts like dogs- and the vector- the Cyclops - as well as the environment -surface water- makes the mission of total eradication of Dracunculus Medinensis in Sudan very difficult.\n\nNo many studies in the literature regarding the water contamination with the Cyclops or the larvae of Dracunculus specially in Sudan have been found. This study aimed to detect and identify Cyclops in Well’s water, Jabal Awliya locality, Khartoum state, Sudan 2021.\n\n\nMethods\n\nThe study was approved by the Research Ethics Committee of the University of Bahri - Faculty of Medicine. Administrative approval from the authority of water, Jabal Al Awliya locality, Khartoum was obtained. Data were aligned anonymously and held with a high level of confidentiality.\n\nThis was a cross sectional study held in the Jabal Awliya locality which is located in Khartoum state, Sudan from 2021-2022. The locality is one of the seven localities in the state which is considered as the main gate for the refugees from South Sudan, Chad and Ethiopia where Dracunculus medinensis has not been eradicated yet. The population is 274,321.\n\nThe main water source in Jabal Awliya is wells water. The design of the well is illustrated in (Figure 1). The water from wells passes into treatment stations then to the tanks. From tanks water passes to the houses to be available for the household use in form of tap water.\n\nThe total number of samples was 264. Samples were collected from wells. Post well water was sampled also. For each area supplied by a certain well a single house was chosen using non-random sampling (convenient) due to unavailability of sufficient data regarding the houses lists. Tap water was sampled in three clean tubes cautiously.\n\nAll the samples were transferred to the laboratory of The University of Khartoum, Faculty of health. Firstly, a light microscope (Olympus BX compound microscope) manufactured by New York Microscope Company (OLBX40F-PATH-40/R) was used to detect the cyclops. Then Cyclopidae were isolated by passing the water samples through special- mesh sieves. All specimens were preserved in ethanol (96%).\n\nThe dissection and identification were done in the laboratory of The University of Khartoum, Faculty of health. Only female specimens were selected for identification; these were carefully transferred with fine dissecting pins from the petri-dish to a drop of water-free glycerine manufactured by Merck KGaA, Damstadt, Germany (catalogue No. 104057) on a glass slide. For identification of cyclopoid copepods, the fine structures of the antennary basipodite segment, the 4th pair of legs, 5th pair of legs and maxillulary palp were of paramount importance. The specimens were placed on the dorsal side with a drop of glycerine; the abdomen was separated from the rest of the body with one dissecting pin. The tools used for preparation of the samples are illustrated in Figure 2.\n\nEach specimen was cut between the thoracic somites three and four, and then the P4 was separated. The abdomen was placed on the dorsal side with the ventral side facing upwards, in order that the 5th pair of legs, the genital somite with the receptaculum seminis and the furcal rami with setae, could be observed. The first antennules were also separated. The 4th thoracic segment was separated from the abdomen of the specimen and placed with the caudal site facing upwards in order that the 4th pair of legs and the uniting lamella can be seen (Idris and Mohamed 2015). Examination of species was done by an Olympus BX 50 compound microscope.\n\nThe identification of the genus Mesocyclops was mainly done by using “The Guides of the Identification of the Microinvertebrates of the Continental waters of the World” (Holyńska et al. 2003).\n\n\nResults\n\nA total of 264 samples were collected, 132 from well’s water and 132 from post wells water sources. Well’s water contamination with cyclops was found to be 2.4%, while post well’s contamination was found to be 8.9%. The species reported by microscopy were: Afrocyclops, Mesocyclops and Microcyclops. Significant statistical difference between well’s and post well’s contamination was found (p-value 0.02) (Table 1).\n\nAnother sample that was collected from Jabal Awliya dam showed contamination with mesocyclops. Pictures for the microscopic identification of the mesocyclops are illustrated in Figure 3 and 4.\n\n\nDiscussion\n\nThe Guinea worm eradication program has been firstly established in 1992. Since that time great efforts are being done in order to eradicate Dracunculiasis as well as prevent the reemergence of the disease. Total eradication has been achieved in North Sudan since 2013. The South Sudanese health minister declared that Guinea worm transmission had ended within the country in March 2018 at the Carter Center. The disease has a 12-month life cycle, and the most recent case was documented 15 months ago (Carter, 2018). Unfortunately reemergence has occurred. The number of human cases has been in the double digits for the previous eight years (54 in 2019 and 27 human cases in 2020). These human cases have been documented in six nations: Angola (1 case), Chad (12 cases), Ethiopia (11 cases), Mali (1 case), South Sudan (1 case), and Cameroon (1 case). They were probably imported from Chad. Many factors raise the risk of importing new cases from the previously mentioned countries. First of all being surrounded with the majority of the endemic countries. Besides that, people of those countries suffer from poor socio-economic status as well as an unstable political situation. As a result of these factors, many people travel to north Sudan, particularly Jabal Awliya territory, which has become a displaced person camp amid the Moment Sudanese Respectful War, lodging more than 100,000 tenants. It got to be a gate way for refugees suffers poor socio-economy, lack of basic health services, high levels of illiteracy leading to malpractices (The March of the Green Flag 1995). What makes travel of refugees easier is that the boarders of Sudan are open and not well secured. The presence of the vector (cyclops) and the sources of infection (refugees and dogs) within the same area creates a risk of reemergence of Guinea worm infection in Sudan.\n\nOne of the crucial precautions that must be taken to stop the Guinea worm transmission cycle from being completed is water safety. Since 2013 the surveillance regarding presence of the vector, as well as the infective agent, has been halted. This study is a part of the surveillance that should be kept specially in this locality aiming to assess the safety of water sources regarding Guinea worm transmission.\n\nThe detection and identification of the copepods - one of the important hosts that transmit dracunculus medinensis - in drinking water is one of the corner stones of the eradication process. In the present study, water from different levels (before and after treatment) as well as Jabal Awliya dam has been assessed microscopically.\n\nThe microscopy results showed that well’s water contamination with copepods was 2.4%, while post well’s contamination was 8.9%. The identified genera are Mesocyclops, Microcyclops and Afrocyclops. On the other hand, samples from the Jabal Awliya dam showed contamination with mesocyclops. Both Mesocyclops and Microcyclops act as vectors for Guinea worm as reported by Johnson (1990). The presence of those types of copepods in drinking water creates a suitable environment for Guinea worm. In other words, whenever worms find their way to water sources, the life cycle will be completed and hence there will be a reemergence of the disease. Copepods are very sensitive for water treatment with simple methods including chlorination, so the detection of such crustaceans in drinking water is an indicator for inefficient treatment. A noteworthy measurable distinction between well’s and post well’s contamination was found (p-value 0.02). The nonappearance of clear water treatment system can be a conceivable clarification for the high rate of contamination of the post-well water. A high species diversity of Cyclopidae was discovered in a subsequent study carried out in Nigeria in 2020 by Yijun Ni et al. pointing to investigate the species diversity and distribution of copepods in freshwater habitats using a mitochondrial cytochrome c oxidase subunit I marker. From the Cyclopidae, 15 populations contained five Tropocyclops species, five Mesocyclops species, and two Thermocyclops species. Numerous conceivable reasons can be considered as clarification of this results difference. First of all, the difference in the water sources from which samples were collected. On the other hand, the variability of water treatment systems used in different countries could be a possible reason. Moreover, the diversity in temperature, humidity, PH and collection seasons can be a coherent clarification for contamination contrast. Lastly, the utilization of molecular techniques by Yijun Ni et al. for distinguishing proof of copepods can be considered as a conceivable cause for this contrast (Yijun Ni et al., 2020).\n\nThe high percentage of post-well contamination indicates a high chance of infection, since the presence of the vector raises the transmission potential of any infectious disease. In case of contamination of water with the fecal material of infected animals, larvae will find their way to the vector (Cyclops). Completion of the worm life cycle will occur whenever people get access to this water. The mission of Guinea worm infection elimination will be unachievable in the presence of high transmission potential. On the other hand, eradication of dracuncluasis as a global goal will be impeded.\n\nA total of 816 water samples from wells, streams, boreholes, and rainfall in the research areas were gathered for a different study conducted by Simon-Oke, Afolabi, and Obimakinde in Nigeria. About 10% of the samples were reportedly contaminated with Dracunculus medinensis and Enterobius vermicularis. This percentage concerns the larvae stage of Dracunculus medinensis which is the infectious stage to the human being (Simon-Oke et al., 2020). In comparison with the current study, a higher chance of illness spread was found in Nigeria at the time of the mentioned study. The contrast between the two studies can be clarified in a way similar to the study by Yijun Ni et al.\n\n\nConclusion\n\nIn conclusion, it is apparent that well’s water as well as dam water are contaminated with copepods (Afro-, Meso- and Microcyclops). Hence Residents in the research regions are at hazard for Dracunculus infection whenever larvae make their way to the water sources since mesocyclops and microcyclops are recognized Guinea worm vectors.\n\nSince no apparent water treatment system has been found in the area under study, it is crucial that water sources in this area be treated before consumption in order to reduce the risk of illness. In addition, since some wells are not well structured and not protected, dogs - one of the animal hosts - have easy access to them. Moreover, rain can drift directly to wells carrying fecal materials and contaminated soil. So these wells should be restructured and covered carefully.\n\nAwareness of the worm cycle as well as the disease should be raised in order to ensure that the larvae from the hosts will not be carried to water sources.",
"appendix": "Data availability\n\nZenodo: Detection and identification of cyclops in well's water, Jabal Awlyia locality, Khartoum, Sudan, 2021. https://doi.org/10.5281/zenodo.7096432 (Moustafa et al. 2021).\n\nThis project contains the following underlying data:\n\n• athar data English.xlsx (This is an excel sheet summarizing the results of microscopic identification of the water samples collected from different sources included in this study).\n\n• Figure 3 mesocyclops under microscopy.tiff (this is a picture of the mesocyclops positive water sample).\n\n• Figure 4 mesocyclops under microscopy.tiff (this is a picture of the mesocyclops positive water sample).\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).\n\n\nAcknowledgments\n\nOur gratitude extends to Prof Saada Mohamed Nour and Dr. Gihan Mahmoud Idris for their invaluable advice, insightful comments and continuous support. We would like to thank Carter center specifically Dr. Mazin for providing our study with all the needed data regarding the previous efforts done in Guinea worm eradication.\n\n\nReferences\n\nArora DR: Medical parasitology fourth edition.\n\nBimi L, Freeman AR, Eberhard ML, et al.: Differentiating Dracunculusmedinensis from D. insignis, by the sequence analysis of the 18S rRNA gene. Ann. Trop. Med. Parasitol. 2005; 99(5): 511–517. Publisher Full Text\n\nCairncross S, Tayeh A: Guinea worm and water supply in Kordofan, Sudan. J. Inst. Water Environ. Management. 1988; 2(3): 268–274. Publisher Full Text\n\nChristen R: Identifications of pathogens-a bioinformatic point of view. Curr. Opin. Biotechnol. 2008; 19(3): 266–273. PubMed Abstract | Publisher Full Text\n\nElsasser SC, Floyd R, Hebert PD, et al.: Species identification of North American guinea worms (Nematoda: Dracunculus) with DNA barcoding. Mol. Ecol. Resour. 2009; 9(3): 707–712. PubMed Abstract | Publisher Full Text\n\nHołyńska M, Reid JW, Ueda H: Genus MesocyclopsSars, 1914.Ueda H, Reid JW, editors. Guides to the Identification of the Microinvertebrates of the Continental Waters of the World, vol. 20. Copepoda: Cyclopoida. Genera Mesocyclops and Thermocyclops. Leiden:Backhuys Publishers;2003; pp. 12–213.\n\nHopkins DR, Withers PC: Sudan's war and eradication of dracunculiasis. Lancet. 2002; 360: s21–s22. PubMed Abstract | Publisher Full Text\n\nIdris GM, Mohamed EE: Taxonomy and geographical distribution of freshwater Cyclopidae (Crustacea:Copepoda) of the Sudan. Sudan J. Sci. 2015.\n\nIlegbodu VA, Christensen BL, Wise RA, et al.: Sources of drinking water supply and transmission of guinea worm disease in Nigeria. Ann. Trop. Med. Parasitol. 1987; 81(6): 713–718. PubMed Abstract | Publisher Full Text\n\nJewsbury JM, Imevbore AMA: Smaller dam health studies. Parasitol. Today. 1988; 4(2): 57–59. Publisher Full Text\n\nJohnson M: Copepod vectors of Guinea worm: A review of West African records , and a local scale study relevant to eradication programmes. Nigerian J. Parasitol. 1990; 9(11): 33–39.\n\nMinistry of Health, Global 2000: Guinea Worm Eradication Programme; National Search Summary 1989. Accra:Ministry of Health;1990.\n\nMoustafa A, Nour S, Idris G, et al.:Detection and identification of cyclops in well's water, Jabal Awlyia locality, Khartoum, Sudan, 2021. [Dataset].2022. Publisher Full Text\n\nNi Y, Ebido CC, Odii EC, et al.: Phylogeography and genetic diversity of the copepod family Cyclopidae (Crustacea: Cyclopoida) from freshwater ecosystems of Southeast Nigeria.2020; 1–11.\n\nRuiz-Tiben E, Hopkins DR: Dracunculasis (Guinea worm disease) eradication. Adv. Parasitol. 2006; 61: 275–309. Publisher Full Text\n\nSimon-Oke IA, Afolabi OJ, Obimakinde ET: The Journal of Basic and Applied Zoology.2020.\n\nSobati H: Epidemiological Study of Parasitic Infections in Bumusa Island, Hormozgan. Iran. J. Parasitol. 2016; Vol. 15(No. 3): pp.425–434. Tehran. PubMed Abstract | Publisher Full Text\n\nThe March of the Green Flag. Spine. May 1995; 11(2): 78. Retrieved 2011-03-29.\n\nThiele EA, Eberhard ML, Cotton JA, et al.: Population genetic analysis OF Chadian Guinea worms reveals that human and non-human hosts share common parasite populations. PLoS Negl. Trop. Dis. 2018; 12(10): e0006747. PubMed Abstract | Publisher Full Text"
}
|
[
{
"id": "158901",
"date": "06 Feb 2023",
"name": "Carlos J. Chaccour",
"expertise": [
"Reviewer Expertise Global health",
"epidemiology",
"public health",
"internal medicine",
"infectious diseases"
],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis is a descriptive study showing the prevalence of copepods in drinking water sources in the Jabal Awliya locality of Sudan, were a large number of refugees from South Sudan have been displaced. The authors highlight the key overlap between vector presence and potentially infected migrants and call for action to prevent reinstalment of local transmission. This appropriately raises concerns around the local receptivity provided by the presence of copepods and vulnerability given my the migrant human/dog population.\nGeneral\nThe text requires thorough editorial review for grammar and readability.\n\nSome spelling mistakes/typos as well: country “boarders” vs borders.\n\nSpecies names should be in italics throughout the manuscript.\n\nSome references are provided as a hyperlink to the website (just flagging this for editorial purposes)\nAbstract\nBackground. The cyclops is not spread by drinking water. The parasite is.\n“Residents are at hazard”. I would suggest emphasizing the environmental receptivity provided by the presence of the vector and the regional vulnerability due to migration of potentially infected humans and dogs through porous borders.\nIntroduction\nParagraph 1. “The cycle is completed by the parasite migrating through the victim's subcutaneous tissues after ingestion of the infected stage, which causes excruciating pain, especially in the joints” Dracunculiasis is generally described as asymptomatic during parasite migration with pain only appearing with blistering and parasite emergence.\nParagraph 2. Grammar. “Elimination of 98% of the estimated 3.5 million cases of dracunculiasis” Should this not be a reduction of cases?\nThe numbers provided are off (98% of 3.5 million), it is actually more, cases are in low double digits (>99.99%)\nThe references provided are somewhat old (2002) and endemicity levels do not reflect the current status (e.g., 20 endemic countries are mentioned) See latest numbers here.\nMethods\nMethods are described with enough detail to allow replication and seem appropriate for the described objectives. Additional details such as sampling timeframe and whether both sources were sampled concurrently are needed for an appropriate interpretation of the difference between the two water sources, this however, does not affect the main objective which is flagging the overlap between the vector and migrant population from an area with active cases.\n\nThe results are presented in a succinct and clear manner.\nDiscussion\nGuinea worm cases have been documented in South Sudan in 2021 and 2022 here.\nThere is some redundancy with the results section as these are restated.\nThe authors mention differences in the collection season between the well and post well water sources. I would encourage including this information in the methods to allow for a better interpretation of the results. Were samples collected concurrently? In what dates/timeframe?\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? Partly",
"responses": []
},
{
"id": "204493",
"date": "25 Sep 2023",
"name": "Philip Downs",
"expertise": [
"Reviewer Expertise My area of expertise if in dracuncluliasis eradication programs as well as other neglected tropical diseases. I feel very well qualified to determine the acedemic merit of this type of study."
],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nStudy aimed to identify and document the presence of copepods in dam and well water in the Abal Awliya locality Khartoum state. The results indicate that Guinea worm could potentially be reintroduced into the area if wells and dams were contaminated by a person infected with an emerging adult worm. Study provides recommendations to prevent reintroduction in the state by advocating for water treatment and improvement of wells and dams to be better protected from infected animal and human hosts.\nAbstract\nResults: There are 13,000 species of copepods – 2,800 which live in fresh. Each of the three groupings - Afrocyclops, Mesocyclops and Microscyclops - would be consider a genus not a species\nEditorial:\nCareful editorial review is needed for grammar and readability.\n\nInstead of using the term “cyclops” suggest changing it to “copepod”. Copepod is more commonly used in scientific journals.\n\nInstead of “wells water” or “well’s water”, it should be changed to “well water”.\n\nAbstract, Background. Instead of “This study aimed to detect and identify Cyclops in wells water, Jabal Awilyalocality Khartoum State, Sudan 2021”, it should be changed to “This study aimed to detect and identify copepods in well water, in the Jaba Awilya locality of Khartoum State, Sudan, during 2021-2022.”\n\nResults: Not clear what is meant by “post wells water sources”. Is that the same as dam water?\n\nWould it be more accurate to describ the water sources as \"wells\" or \"cisterns\"? Wells typically are understood to be narrow shafts, but the study implies animals could have easy access to the water at the bootom of the well. This needs to be clarified.\n\nChange “at hazard” to “at risk”\nIntroduction:\n1st paragraph. 4th sentence, Change “embryos” to “larvae”\n\n1st paragraph, 5th sentence. Change the sentence – as it reads right now it implies that the worm parasite ingests the in third stage infected copepod.\n\n1st paragraph, 6th sentence. Change “pops out” to “slowly emerges”.\n\n2nd paragraph. Author needs to reference a more recent article as the majority of cases are no longer in South Sudan. Suggest referencing WER9820-205-224\n\nhttps://iris.who.int/bitstream/handle/10665/367924/WER9820-205-224.pdf?sequence=1\n\nThird paragraph. Technically the copepods (cyclops) do not carry the worms, but the larvae which molt to become third stage infective larvae. Last paragraph. Change “No many” to “not many”.\nMethods\nClarify whether samples were collected from single households or whether the wells were public; i.e. serving multiple households.\n\nWere samples also taken from dams? In the conclusion it states that dam water was contaminated with copepods, but not clear from methods whether samples were collected from dams. Temporal data (month) that samples were collected and the depths of the wells would be informative.\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? Yes",
"responses": []
}
] | 1
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https://f1000research.com/articles/11-1577
|
https://f1000research.com/articles/10-925/v1
|
15 Sep 21
|
{
"type": "Research Article",
"title": "Environmental perceptions of global business travel by Swiss companies in the Zurich airport region",
"authors": [
"Ignacio Echeverria Arrondo",
"Bert Wolfs",
"Bert Wolfs"
],
"abstract": "Background: This article presents findings from research conducted before the coronavirus disease 2019 (COVID-19) pandemic on companies located in the Zurich airport region of Switzerland, regarding the needs for global business travel and its impacts. Methods: The study involved a mixed methods approach. Five hypotheses were tested using inferential statistics on data obtained from pre-tested closed questions in a web-based survey. Deeper context was explored through an interview-based case-study conducted at a Swiss pharma company. Results: Supporting alternative hypothesis 3 (Ha(3)), a significant positive relationship was found between travel frequency and business growth, F(1, 100) = 11.31, p = 0.0011. Supporting Ha(4), corporate culture had a significant positive relationship with business travel frequency (F(1, 100) = 15.50, p = 0.0002) and average trip length (F(1, 100) = 6.39, p = 0.01). And thirdly supporting Ha(5), corporate social responsibility had a significant relationship with global business travel (91%). Ho(2) and Ho(3) were accepted. The case study found that smart corporate travel policies and regulations should be instantiated to enhance our environment, which would also benefit employee wellbeing. Travel can be reduced significantly despite being demonstrated that physical co-presence is important for building trust. The case study suggests tools to support the monitoring and management of global business travel by organizations. Conclusions: COVID-19 has impacted travel for business significantly, and future research will be necessary to assess its impact. The article explores the ongoing research in this area, and several relevant implications are proposed for future leaders. The case study found willingness to pay both corporate and individual green taxes, and a deficiency in corporate communication around the environment. Business travel is needed to build trust; however, it can be reduced.",
"keywords": [
"Switzerland. Business travel. Covid-19. Environmental awareness. Mobility. Corporate Culture. Leadership."
],
"content": "Introduction\n\nPrior to the coronavirus disease 2019 (COVID-19) pandemic, global air travel had never been more popular. Industry projections expected the industry to serve 20.9 billion air passengers per year by 2040, due to cheaper airfares and increasing flight availability (Airports Council International, 2018; International Air Transport Association, 2018). Connectivity via flight had never been better. However, this connectivity comes with several downsides for the global community; the best-known of which are greenhouse gas emissions. In Switzerland, carbon dioxide (CO2) emissions from air transportation are 4% of the country’s total emissions, of which about a third can be attributed to business travelers (Flughafen Zürich Ag., 2018).\n\nThe proliferation of international business in the era of the pre-COVID-19 global economy generated constant growth in the number of individuals who engaged in long-distance travel for work-related activities (Gustafson, 2012). Derudder and Witlox (2016) typifies the literature on this growth. In their discussion of mobility and hypermobility, such travel is expected from employees as part of their day to day working lives. It becomes increasingly common as people rise to more senior positions and is supported by a culture which views travel as an expectation, and indeed a privilege to which employees should aspire.\n\nHowever, others such as Cohen, Hanna, and Gössling (2018) take a more critical stance on the prevalence of travel in business (Gössling, Hanna, Higham, Cohen, & Hopkins, 2019). For many employees, travel is a burden imposed from above, with significant proven health risks and many personal downsides (DeFrank, Konopaske, & Ivancevich, 2000; Westman & Etzion, 2002). Employees who feel this way face challenges in their lack of enthusiasm for travel, particularly as they climb the corporate ladder. The study on which this paper is based, conducted prior to the emergence of COVID-19, began when the researcher asked himself: why do we travel so much for business? Is it because we must for business-reasons? Or is it mostly because of an established culture of travel? What if we do not?\n\nAs well as employee wellbeing, the environment provides a pressing rationale for this reconsideration. Mr. Antonio Guterres (Secretary-General, United Nations) stated in the COP25 opening ceremony at the Madrid Summit in December 2019, that climate change is now a threat to life across the world. Alongside intergovernmental organizations, climate activists have issued a clarion call for actions such as demanding an end to fossil fuel subsidies and the implementation of restrictions on carbon-emitting activities from worldwide leaders, rather than relying on passive target setting. Their demands, along with the aspirations of COP21, are near-impossible to reconcile with expectations of continued growth in air transport and business travel.\n\nThe reasons given for business travel have been the subject of study for several years. Above all, the context is one in which current business leaders and executives have a strong preference for face-to-face meetings over technological alternatives (Koyen, 2009; Harvard Business Review, 2009). There are two areas in which that preference is to an extent backed up by research: the transfer of knowledge, and trust-building. Both are better accomplished in some circumstances by face-to-face communication, and the ensuing associated travel, than through any of the available virtual alternatives (Tani M, 2005; Welch, Welch, & Worm, 2007).\n\nBut the environmental load, and the negative impacts of extensive travel on employee health and retention, are both pressing. The gap in the literature linking the environment with corporate social responsibility (CSR) was clearly identified as early as 1994: “this concern for the environment serves as a new, unifying theme for the study of international business, which will no doubt continue to deepen and grow in importance” (Wright & Ricks, 1994, p. 699). In some areas it has received attention – there are a range of studies on resource use, production-based pollution, and the range of responses firms can take from internal policies to engagement with regulatory bodies. These were often prompted by large-scale and widely reported disasters, along with responses to governance issues en-vogue at various historical moments. But awareness remains sparse and further studies are needed to highlight options that might work in particular settings (Kolk, 2016).\n\nWhile the COVID-19 pandemic has seen a huge drop-off in air travel, there are indications that many businesses intend to resume travel in the near-term. However, as has been argued in many fields, this may provide a one-off opportunity to reset travel on a more sustainable footing, and to break the hold that travel has over business culture (Koonin, 2020; Hepburn et al., 2020; Kuzemko et al., 2020). The current normal is remote, and in some areas, it is allowing equivalent if not superior functionality to in-person communication (Clopper, Baccei, & Sel, 2020). The potential for an increased use of technology to replace travel has been building for a decade or more (Julsrud, Denstadli, & Hjorthol, 2014). It may now be realized.\n\nThe study reported below, conducted prior to COVID-19, demonstrates that not all business travel is essential, and will add to the evidence-base that argues a more targeted and specific approach to business travel would benefit employees, the environment, and businesses in the future (Poom, Orru, & Ahas, 2017). Leaders of the future should de-normalize travel both in how they act and the policies they engage in for their employees.\n\nSwitzerland is a small landlocked country located in central Europe, with a population of 8.5 million people, as of the 2018 census (Federal Statistical Office, 2018). Switzerland receives almost double the number of travelers that would be anticipated from its expenditure (Bripi, 2019). This indicates that Swiss companies attract business travelers disproportionately to their size, and this can be applied doubly so in the Zurich area. The study focuses on the Zurich airport region in Switzerland; a population of around 129,329 inhabitants in the following communities: Bassersdorf, Oberglatt, Rümlang, Opfikon-Glattbrugg, Wallisellen, Wangen-Brüttisellen, Dietlikon, Dübendorf, Nürensdorf and Kloten. Zurich Airport (2018) states that 31 million passengers used Zurich airport, of which 27% (or 8.37 million passengers) were international business travelers.\n\nHow business travel is viewed within a country varies not just with frequency but also with the different perspectives that people bring to bear on it. Switzerland is a country where the people are widely known to be environmentally friendly, outdoor sport-oriented and nature lovers. This is reflected in Switzerland being the first country to hold a ballot on implementing a green economy (in September 2016). Research demonstrates the consequential value that the Swiss place on their time; for instance, Bates, Bierlaire, Abay, Axhausen, and König (2006) examined how much one hour of traveling was worth to people in different countries within Europe. They found that Swiss citizens place a higher value on their time than any other European citizens do. In Swiss culture, free time and traveling are signs of status – with first class travel and particularly air travel acting as additional signifiers.\n\nTwo studies provide insight into how the Swiss view air travel and business travel. Firstly, Bruderer Enzler (2017) provides a short account of private air travel in Switzerland. Of interest is the brief examination of “attitudinal predictors”, which analyze behavior on a range of factors based on surveying undertaken in 2007. Of most relevance here was the finding that while those who voted for the Green Party of Switzerland, caused lower emissions overall, they flew nearly as often as the rest of the population. Hinnen, Hille, and Wittmer (2017) found that a significant proportion of passengers might be willing to pay extra for “green” products associated with air travel, such as organic on-board food or carbon offsets.\n\nThe main aim of the study was to help the author understand two simple questions: why do we travel so much for business. Is it because we have to, or because we need to? To answer these, a study was undertaken using both quantitative and qualitative research methods focused in businesses within the Zurich airport area in Switzerland. The study will contribute a detailed case study to the literature, with a greater focus on the intersection between environmental awareness and business travel, than previously published work.\n\n\nMethods\n\nThis study followed a mixed-methods approach, utilizing both a survey and a detailed case study. This section will outline the main methodological features of the study and give detail as to the characteristics of both the survey respondents and case study. Hypotheses were developed based on secondary literature research before the study was undertaken and are reported in the text below where appropriate. All study materials can be found as extended data (Echeverria-Arrondo & Wolfs, 2021).\n\nIn total, five hypotheses were tested quantitatively using pre-tested closed questions in a web-based survey, which was collecting data from September to December 2019.\n\n• Ha(1) “As environmental awareness (from the corporate) increases, business travel decreases”.\n\n• Ha(2) “As environmental awareness (from the individual) increases, business travel decreases”.\n\n• Ha(3) “There is a significant positive relationship between global business travel and business growth”.\n\n• Ha(4) “Corporate culture is the predominant reason business travel is undertaken as frequently as it is”.\n\n• Ha(5) “As corporate social responsibilities policies linked to environmental awareness (from the corporate) increases, business travel decreases”.\n\nInformation regarding the operationalization of the three major constructs, along with the evaluation of specific hypotheses, can be seen in Table 1. Each construct is listed along with any relevant variables, and the specific questionnaire items that form one. The six columns on the right denote which of the five specified hypotheses are being tested.\n\nThe questionnaire designed by the researcher was tested for validity and reliability using Qualtrics test survey tool. Pilot testing was undertaken with 10 people in August 2019. The criteria for selection of these 10 people was to hold a role in a company involving traveling, two with native English, and the rest German. They were asked beforehand to check their willingness and availability for participation. They were asked to highlight any unclear questions and to provide overall feedback which was used to improve the final survey. This was especially important as the survey was bilingual, in German and English, with the researcher not a native speaker of either (see Appendices A and B). The participants also timed how long the test took to complete, in order to assess whether it needed to be shorter or any questions amended. The researcher made some corrections based on the feedback to make the questionnaire clearer avoiding ambiguity and corrected the German language.\n\nThe study is based on the region surrounding Zurich airport, in which the latest research available (from 2014) states that there are 118,626 people employed (Flughafen Zürich Ag., 2014). Informal consultations with local human resources (HR) managers resulted in an estimate that 30 to 40 percent of all jobs within the Zurich Airport region involve global business travel, and thus might fall under consideration for this study. Therefore, the appropriate total population size for this study is 36,000 individuals. Mugenda and Mugenda (2012) posited that where a research population is large, the recommended minimum sample size is 384. This is predicated on the assumption that the population is normally distributed, and the degree of confidence is 95%, at a significance level of 5%.\n\nThe study utilized multistage sampling, in which clusters of the overall population are selected and then random samples drawn from each of those clusters. This allows a good balance of cost, convenience and accuracy compared to alternative sampling methodologies (Gall, Gall & Borg, 2007). In this case, clusters were formed of companies with which the researcher had a pre-existing relationship. A random sample of their employees was then taken for the survey. Participants were contacted, either by phone or by email, to gain their consent to invite them to take part in the survey.\n\nThe survey was sent to target 400 respondents in September 2019 and conducted through Qualtrics software, Version 2019 to collect the data and perform quantitative statistical analysis. Google Forms or SurveyMonkey could have been used as open access survey alternatives, paired with Apache OpenOffice for quantitative analysis.\n\nFor each question of the survey, responses were coded numerically along a seven-point Likert scale ranging from “very strongly disagree” (coded as -3) to “very strongly agree” (+3), with “neither agree nor disagree” as the neutral option (0). This allowed examination of the correlation between different responses. Then Pearson’s correlation co-efficient was utilized to draw conclusions about the strength of the correlation. Standard deviations provided information on the breadth of opinion. Regression analysis was performed, where warranted, to illustrate the relationship between the variables. Analysis of variance (ANOVA) was used in inferential tests when determining the strength of influence that the independent variables had on the dependent variable.\n\nA case study, the interviews for which were carried out between August and September 2019 at a Swiss pharma company in the region provided further in-depth understanding of the reasoning and intentions behind the business travel. Ms. Silvana Micheli, a Swiss citizen native in German, Swiss-German and very high level of the English language, was trained by the researcher to perform the interviews on his behalf to avoid any bias or influencing the interviewees. Ms. Silvana Micheli is a Learning and Development professional in the pharma industry.\n\nIn the organization, between 30 and 40 percent of all roles involve traveling. Overall, 15 employees were selected for the case study, a number which was felt to be sufficient for additional detail without risking data saturation. They were identified starting from the nominal description: gender, age, role grade and type of role and department. Only those with a role involving traveling were included. They were intentionally chosen to represent a broad scale of seniority and approached by email for their consent to participate in August 2019. At that time, they were informed of the nature of the study and the topic area, but not the research.\n\nAn NDA (non-disclosure agreement) was issued and signed by representatives from the pharma company. The male researcher countersigned it to preserve the anonymity of the company and its employees. The data collected has been stored at SBS Swiss Business School safely and will be kept for five years. After this period, under the terms of the NDA the material including audio recordings of the interviews will be destroyed.\n\nAn interview format was implemented with participants and questions were based upon the structure of the survey questionnaire to enable meaningful comparison of results. This gave the interviews a clear structure while allowing the possibility of extended answers and follow up questions as appropriate. Sessions were 45 minutes long, one on one, took place in the offices of the pharma company, and audio was recorded for the purpose of this research study only. The interviewer also took notes, manually writing down the answers to the interview questions. No repeat interviews were necessary. No participant refused to take part. Transcripts were not made available to participants. At the beginning of each interview the interviewer read the consent for audio recording and provided two copies to be signed. The interview audio analysis was part of a broader study than reported here.\n\nAn ethics declaration form was submitted to and approved by SBS Swiss Business School Human Resources Ethics Committee (approval number: 642). Additionally, a research panel consisting of a supervisor assigned by SBS Swiss Business School and industry specialists, examined the quantitative and qualitative surveys and this research proposal, before giving ethical permission for the study.\n\n\nResults\n\nIn total, 104 completed questionnaires were received in response to the quantitative study (Echeverria-Arrondo & Wolfs, 2021). This was a response rate of 26%, which given that rates as low as under 10% are not uncommon with web surveys, was reasonable. However, future researchers may want to take account of the advice in van Mol (2015) and provide repeated digital reminders for potential participants. To prevent lengthening the quantitative survey, which might have further impacted the response rate, demographic data was not collected. The age range of the respondents is estimated by the researcher to have been between 25 and 60 years old. Around 30-40% female and 60-70% male.\n\nFor the case study, four of the 15 participants were female, and 11 were male (26%, 74% male). Their ages ranged from 25 to 65: 25-34: 3 (20%), 35-44: 4 (27%), 45-54: 6 (40%), 55-65: 2 (13%).\n\nTravel time and trip length varied markedly among both survey respondents and case study participants, as is shown in Figure 1. A Pearson’s Chi-squared test of independence (with Yates’ continuity correction) between travel frequency and trip length was not significant, χ2(1) = 3.31e-31, p = 1. It was therefore acceptable to use them as independent variables for inferential statistics.\n\nFollowing Ham, Mrčela, and Horvat (2016) this study defined environmental awareness not only as a knowledge of environmental issues, but also a willingness to act on climate science as a corporation and as an individual. In business, that tends to be financial remuneration to the global citizenry in some form of green tax or financial offsetting of carbon footprint. There were thus two question sets which, taken together, illustrate ‘environmental awareness’.\n\nRegarding knowledge the questions asked were:\n\n• Q9) “I understand the greenhouse effect, its causes, and its consequences.”\n\n• Q10) “I understand the greenhouse gas emissions caused by an aircraft.”\n\n• Q11) “I understand the consequences of the global warming.”\n\nTable 2 displays the correlation matrix for the three “climate science knowledge” questions. The three questions were highly positively correlated with one another, with Pearson R values ranging from 0.75 to 0.89. Since they are so strongly correlated, they are combined into one overall variable called “climate science knowledge” by averaging them together. Scores on this measure ranged from -0.333 to + 3.00, with a mean of 1.88, and a standard deviation of 0.876.\n\nFor each of the three statements, the percentage of respondents who endorsed the statement (“understand”, “strongly understand”, “very strongly understand”) was over 95%.\n\nAnd regarding willingness to act:\n\n• Q16) “I am willing to pay more, as an individual, when purchasing pollution products and services, through “green taxes”.”\n\n• Q17) “I am willing to pay more, as a corporation, when purchasing pollution products and services, through “green taxes”.”\n\n• Q18) “I am willing to pay (as an individual) a fee to NGOs such as “myClimate”, when purchasing pollution products and services – business related – through off-setting carbon footprint compensation.”\n\nTable 3 displays the correlation matrix for the three social responsibility questions. The three questions were positively correlated with one another, with Pearson R values ranging from 0.58 to 0.65. Since these responses are correlated, they were combined into one overall variable identified as “social responsibility”, by averaging them together. Scores on this measure ranged from -0.333 to + 3.00, with a mean of 1.88, and a standard deviation of 0.876.\n\nThis was not proven from the data collected. There was no significant difference between business travel and wiliness to pay corporate taxation. For ‘more’ and ‘less’ frequent travelers, F(1, 100) = 0.041, p = 0.840. Therefore, the null hypothesis was accepted. No effect was found upon trip length, F(1, 100) = 0.33, p = 0.86, nor was there any interaction between the two business travel variables, F(1, 100) = 1.96, p = 0.165.\n\nAgain, the null hypothesis was accepted here with no statistically significant relationship being found. The analysis did not find a significant difference between more and less frequent travelers, F(1, 100) = 0.001, p = 0.979. The analysis did not find an effect on trip length, F(1, 100) = 0.18, p = 0.68, nor any interaction between the two business travel variables, F(1, 100) = 0.01, p = 0.93.\n\nThere was a noticeable relationship between the variables making up environmental awareness at the corporate level: “climate science knowledge” and “corporate social responsibility”. As shown in Figure 2, a positive correlation was found, Pearson’s product-moment correlation R = 0.309, 95% confidence interval (CI) = 0.124-0.473, t(102) = 3.282, p = 0.0014.\n\nThe next question which needed addressing was: is this relationship affected by business travel? To examine this more closely, the respondents were split into more frequent and less frequent travelers to be plotted in that relationship. This can be seen in Figure 3. For those who tend to travel more frequently, “climate science knowledge” and “social responsibility” are positively related, R = 0.528, 95% CI = 0.274 – 0.713, t(42) = 4.03, p = 0.0002. However, there is no such correlation for those who travel less frequently, Pearson’s product-moment correlation R = 0.160, 95% CI = −0.097 – 0398, t(58) = 1.24, p = 0.221.\n\nThe analysis combined all the variables that were found related to global business travel, here operationalized as travel frequency. The researcher discovered that elements of “corporate culture” were related to “business travel”: liking business travel; and believing business growth comes from travel. It was also found that an interaction of the elements of “environmental awareness” were related to “business travel”: similarly, “science knowledge” and “corporate social responsibility” are related.\n\nHa(3), “there is a significant positive relationship between global business travel and business growth”.\n\nTo test this hypothesis, it was necessary to take both the responses to questions 1 and 8, and then consider the answers on a branching basis. The results of which are displayed in Table 4, with the number of respondents displayed.\n\n• Q1): “traveling for business increases business growth.”\n\n• Q8): “in my experience, business travel has been essential for achieving the results I have.”\n\nA Pearson’s Chi-squared test of independence (with Yates’ continuity correction) was statistically significant, χ2(1) = 5.5283, p = 0.01871. Given that dependence, for each respondent “business growth” was calculated by taking the average of the responses from Questions 1 and 8.\n\nIf global business travel is undertaken partly because people believe it contributes to their business growth, it would be expected that travel frequency be related to business growth. However, it would not necessarily be expected that the variables would be dependent to the extent that it would predict an average trip length. That is, those who travel more frequently do so because they believe it is integral for their business growth, and that trip length should not be related.\n\nA 2 (trip length: shorter, longer) × 2 (travel frequency: less frequent, more frequent) between-subjects ANOVA was conducted to see whether “business growth” varied across the groups. The data showed that there was a significant difference for travel frequency: indeed, those who travel more frequently did rate travel as more important to their business growth.\n\nFigure 4 is a scatterplot showing one blue point for each of the respondents, based on which of the four options they chose for travel frequency and their judgment of how important travel is to their business growth. There is random jitter added to the graph, so that each point is visible. The purple dots show the average number of people at each possible response by their size. The purple line shows the linear relation between the two variables. Analysis of this supports hypothesis 1, as there is a significant and positive correlation. Pearson’s product-moment correlation R = 0.299, 95% CI = 0.113 – 0.465, t(102) = 3.164, p = 0.002053.\n\nThe interviewees all believed that “business travel has been essential for achieving the results” they have; and two thirds of them also endorsed the idea that business travel increases business growth. There were several discussion points which provided an explanation of their rationale, and complicating factors. One interviewee mentioned the problem with regular travel and maintaining office relationships and workload. The idea of balance was mentioned by several respondents. The stresses of business travel were notable, but no consensus emerged from the group regarding its suitability for them as employees.\n\nRegarding virtual teams, one of the interviewees said that to develop a team located in different sites, the sense of belonging to a team and being together developing relationships and trust, is key to success. And face-to-face contact remains by the best means to establish that rapport. However, interviewees supported the idea of Oertig and Buergi (2006) that irregular physical meetings, perhaps only annually, is enough to keep a virtual team working efficiently. Therefore, while face-to-face contact is needed within team-based environments, it can be kept to a minimum without significant drawbacks.\n\nThe overall message from the interviewees, supporting most of the literature mentioned above, is that achieving a balance is the goal to optimize employee and corporate prosperity.\n\nHa(4), “corporate culture is the predominant reason business travel is undertaken as frequently as it is”.\n\nThis was tested quantitatively with two questions:\n\n• Q6) “Video/audioconferencing is preferable to a business trip.”\n\n• Q7) “Information technology (IT) has reduced the use of air travel for business purposes.”\n\nThere was a significant positive correlation between responses to the two questions (see Figure 5), Pearson’s product-moment correlation R = 0.51, 95% CI = 0.282 – 0.592, t(102) = 5.1003, p = 0.000002. The researcher reasoned that respondents from corporations with a meeting culture that emphasizes meeting face-to-face would report greater enjoyment of business travelling. For one, they were hired by the corporation, so they are likely to embody the culture to begin with. Secondly, being in a pro-business-travel meeting culture would ultimately make one more likely to believe they enjoy business travel, as has been argued previously (Bentley, Bloomfield, Davidai, & Ferguson, 2016; Mueller, 2010). The researcher compared the responses to this question against the variable “remote meeting” and found that they were not significantly correlated (see Figure 6), Pearson’s product-moment correlation R = 0.062, 95% CI = −0.133 – 0.253, t(102) = 0.626, p = 0.533. Enjoyment of business travel and a preference for videoconferencing are two separate constructs.\n\nThe interviews gave some further light into this disconnect. It was said that better sharing of the information on how, where, who, and what for, business travel is occurring, would mean that the travel can be consolidated as smart travel and reduce the amount of unnecessary travelling. A problem with this is that people might not be willing to share all the intentions of business travelling. An additional aspect to consider is that training to use appropriate technology is required to make people use them. If the videoconferencing equipment were advanced, secure, and well supported then this would reduce travel for business. Given COVID-19 standards are emerging and gaining broader support in this area (Kagan, Alpert, & Fire, 2020; Weil & Murugesan, 2020).\n\nFor the corporation, interviewees found several benefits for travel. These included: understanding the local market, interaction with affiliates, motivation, better results, build relationships, business growth, generate more business, and trust building. It allows people to build up relationships, make decisions quicker, and avoid the infuriating ‘ping-pong messaging’ often seen in remote communication. Business interactions involve a myriad of nonverbal cues (including body language) and therefore face to face meetings have the advantage of capitalizing on that. Yet for the individual engaging in business travel, the free discussion was predominantly negative. It was voiced that traveling for business is exhausting and that it is not beneficial for everyone; the load on businesspeople with families was mentioned several times, as was health.\n\nHa(5), “as corporate social responsibilities policies linked to environmental awareness (from the corporate) increases, business travel decreases”.\n\nThe survey asked the following question directly:\n\n• Q19) If corporations pursued pro-environment corporate social responsibility policies, would business travel increase or decrease?\n\nOf 102 responses to that question, 93 (91%) responded that business travel would decrease in such a situation, and 9 (9%) responded that it would increase. Therefore, 91% of the respondents endorsed Ha(5) in the hypothetical. A similar result was achieved in the interviews – 14 of the 15 respondents (93%) endorsed the question.\n\nOne of the interviewees indicated that if pro-environment policies were implemented, travel for business would be reduced in the short term only; the corporation would need to keep investing in a pro-environment program. To succeed, it would need to be implemented and embedded as part of the culture of the corporation. Another respondent said that if a company was environmentally friendly with the usage of paper, water waste, recycling, etc., then it must also consider the impact of business travel on global warming. Physically travelling for business purposes needs to be seen not as purely a sunk cost in travel expenditure. It must be measured in terms of environmental impact as part of the values of the corporation. Without a quantified measure such as that, pro-environment policies would tend to be unsustainable in the long run. These kinds of policies only work when supported and believed in by senior leadership.\n\nThe interview brought out an important point regarding CSR policy. Only four of the 15 interviewees knew that they did already have a policy in place. Of these, only two of the four said the policy referred to travel for business, one said the environment, and the other to neither of them. One of them indicated that the company at the focus of the company’s own case study is moving from Scope 2 up to Scope 3. Scope 2 are indirect emissions from the generation of purchased energy (Greenhouse Gas Protocol Corporate Standard (GHGPCS) Value Chain Emission) whereas Scope 3 emissions include all indirect emissions that occur in the value chain of the reporting company, including both upstream and downstream emissions. All four belonged to the more senior group of employees interviewed.\n\nFour of the other interviewees (27%) replied that the company did not have a CSR policy. Seven did not know. Therefore, it can be assumed that related internal information is not distributed in a clear and homogeneous manner within the business for all employees. It would seem evident that internal communications are an area for the business to focus upon in the future. Other literature indicates that this finding is far from unusual (Yue, Men, & Ferguson, 2020), but given comments above about the importance of communication from senior leadership to embed pro-environment policies, it is of note.\n\nIt is also worth noting that travel policies were in place within the company to control expenditure, hotel grade selection, car hire price range, weekend allowances, flight price range (related to business class, economy, or premium economy) depending on the length of the flight, etc., but not related to any environmental concerns or CSR. Again, this is far from unusual.\n\nThere were three items on the questionnaire regarding environmental awareness:\n\n1) “I understand the greenhouse effect, its causes, and its consequences”;\n\n2) “I understand the greenhouse gas emissions caused by an aircraft”; and\n\n3) “I understand the consequences of the global warming”.\n\nResponses to these three items were correlated with responses on the item “I am willing to pay more, as a corporation, when purchasing pollution products and services, through “green taxes”.” The research analyzed the relationship between these knowledge-based variables and willingness to pay both corporate and individual green tax (see Table 5).\n\nUnsurprisingly, the three questions about environmental awareness were highly positively correlated with one another, with R values ranging from 0.75 to 0.89. The correlation with willingness to pay corporate green tax were also positive, and statistically significant, with R values of 0.23, 0.26, and 0.37 (see Table 6).\n\nThe correlations with “willingness to pay individual green tax” were also positive, and statistically significant, with R values of 0.26, 0.30, and 0.31.\n\n\nDiscussion\n\nWhile business travel is a global issue, many of the cultural aspects discussed above have a local element. The study was focused upon the Zurich airport area in Switzerland, a country which is renowned for its environmental awareness. It may be that in other geographical regions, different results would be found which would add to the body of knowledge regarding the relationship between the major constructs of the study.\n\nPerhaps more significantly, the pandemic of COVID-19 has placed a chronological limitation on this study, and indeed any recently conducted study regarding business travel (along with many other aspects of life). While the environmental imperative has not changed, many other influences on business travel and the corporate culture around it have altered markedly since the research was carried out. Therefore, this study will provide a crucial point of comparison for later work looking at the same topic: a clear ‘before’ point to assess the impact of COVID-19 on business travel habits and opinions.\n\nIf the culture of a corporation is pro-business travel, one might expect more travel will be taken and less videoconferencing or remote meetings will be arranged. This is justified by those corporations based on business growth, which as this research has shown, is considered to be facilitated by face-to-face meetings, particularly in the early stages of many business relationships. It has been demonstrated that physical co-presence is important for building trust quickly (Castells, 2010; Faulconbridge, Beaverstock, Derudder, & Witlox, 2009; Lo et al., 2013), as was reported by the interviewees with reference to virtual teams above.\n\nThe interviewees had several inputs of value for a discussion of broader corporate culture:\n\n• Need for CSR policies embedding environment and business travel.\n\n• Sustainability to be part the corporate values.\n\n• Executives to be role models to make the message stronger.\n\n• Internal communication improvement. & clearer strategy/guidance.\n\n• Practice and recognition to become part of the company culture.\n\n• Allocation of a measuring manager (KPI).\n\n• CO2 emissions monitoring and measuring.\n\n• Willingness to pay for CO2 emissions (green taxes)\n\n• In the job description, the travel percentage needs to be defined and indicate how to be measured.\n\n• To be successful developing a remote team it is key the sense of belonging and being together developing trust and relationship.\n\n• Increasing ICT (information and communications technology) tools and training for optimal usage.\n\n• Paying for CO2 emissions.\n\n• The use of electrical cars as a company cars policy.\n\n• Smart traveling awareness and implementation – consolidation.\n\n• Definition of electrical cars as a company car.\n\n• Legal regulations from the local authorities.\n\n• Clearer strategy and corporate guidance.\n\n• General reduction of business travel.\n\n• To be always traveling would be a disadvantage to the organization and subsequently, balance is the goal for the employee and corporate prosperity.\n\nThe overriding sentiment was that these factors must be embedded into business culture. COVID-19 has undoubtedly provided the environment a break from air and noise pollution, which is notable from datasets in the public domain. The pandemic has also done plenty to illustrate just how much people can change their behaviors, when they are highly motivated or forced to do so. Taking up some of the issues listed above would enable corporations to facilitate both a business culture that contributes positively to social and environmental issues, and which takes care of its employees. Traveling for business is hard on the body mentally and physically, as well as time consuming (Bunn & Johnson, 2019; Mäkelä & Kinnunen, 2018). Therefore, it is sensible to conclude that videoconferencing should be used more where the downsides of travel are not offset by significant growth probabilities. There is significant relationship between business growth and business travel as face-to-face meetings are essential to build trust and relationships. However, travel can be significantly reduced, by more than 50% as indicated the interviewee 2, and depending on the purpose of the meeting, it may not be necessary, for instance for standard topics (Bripi, 2019). In matrix organizations once a year face to face meeting is sufficient to run virtual teams (Oertig and Buergi, 2006).\n\nThe case study found the willingness to pay both corporate and individual green taxes was positive and statistically significant. Further replication of the study would be desired to conclusively determine if these significant values could be generalized beyond the study population. Particularly so as Switzerland is renowned for its environmental awareness, which may make the population atypical on a European, and indeed global, level. Nevertheless, the results of the study add to a body of evidence that there is willingness to pay green taxation if the Swiss government were to implement it (Bernhard, 2020; Maxim, Zander, & Patuelli, 2019).\n\nAdditionally, it is evident that corporate communication on environmental issues is currently suboptimal. The case study demonstrated that even in an organization which is making positive moves towards Scope-3 emissions considerations, the communication of that desire within the company, was lacking. A notable absence from the interviews conducted during the case study, were the United Nations 2030 Sustainable Development Goals, which went entirely un-mentioned by participants. Government policies must develop mechanisms to highlight this when implementing CSR regulations (Rela et al., 2020; Pinzone et al., 2019).\n\nWith COVID-19 providing a push towards the utilization of videoconferencing systems, many individuals and corporations are being forced to develop the skillsets needed to make best use of technology. Companies are investing in appropriately secure videoconferencing systems, server, and broadband technologies. Will restrictions on travel be a long-term part of life? Will businesses make the conscious choice to embrace virtual communication where it is most appropriate? Or will business travel return to something like the pre-COVID normal, given time? Fundamental questions like this remain unresolved. Further research will thus be essential to assess the longer-term implications of COVID-19 in this area.\n\n\nData availability\n\nUK Data Service: Environmental Perception of Global Business Travel by Swiss Companies in the Zurich Airport Area, 2019-2020. https://doi.org/10.5255/UKDA-SN-854930 (Echeverria-Arrondo & Wolfs, 2021).\n\nThis project contains the following underlying data:\n\n- Survey individual raw data.xlsx\n\n- Survey master data from Qualtrics.csv\n\n- Case study interview results overview.xlsx\n\n- Case study interview transcripts.pdf\n\nUK Data Service: Environmental Perception of Global Business Travel by Swiss Companies in the Zurich Airport Area, 2019-2020. https://doi.org/10.5255/UKDA-SN-854930 (Echeverria-Arrondo & Wolfs, 2021).\n\nThis project contains the following extended data:\n\n- Case study interview consent form.pdf\n\n- Survey questionnaire in English and German.pdf\n\n- Survey report from Qualtrics 2019.pdf\n\n- Case study interview schedule August - September 2019.pdf\n\n- Case study introduction letter.pdf\n\n- Non-disclosure Agreement for the case study.pdf\n\n- Qualitative questionnaire - case study.pdf\n\n- Chapter 4 from the dissertation - quantitative - research findings and discussion.pdf\n\n- Chapter 5 from the dissertation - qualitative - case study swiss pharma company.pdf\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).",
"appendix": "Acknowledgements\n\nSpecial acknowledgement and gratitude to Bert Wolfs Ph.D., Academic Dean at SBS Swiss Business School, for his invaluable guidance and support. I also highlight my gratitude to my partner Silvana Micheli for her support. Both authorised me for their name to be mentioned on the publication.\n\n\nReferences\n\nAguilera A: Business travel and mobile workers. Transportation Research Part A: Policy and Practice. 2008; 42(8): 1109–1116.\n\nAirports Council International [ACI]: ACI’s world airport traffic forecast reveals emerging and developing economic will drive global growth [Press release].2018, November 1. Reference Source\n\nBates JJ, Bierlaire M, Abay G, et al.: Swiss value of travel time savings . 2006. Publisher Full Text\n\nBentley JW, Bloomfield RJ, Davidai S, et al.: Drinking Your Own Kool-Aid: The Role of Beliefs, Belief-Revision, and Meetings in Persuasion.AAA; 2016, August.\n\nBernhard L: The 2019 Swiss federal elections: the rise of the green tide. West European Politics. 2020; 43(6): 1339–1349. Publisher Full Text\n\nBripi F: Business travels, trade, and multinational firms. Bank of Italy. 2019; 0(0), 36.\n\nBruderer Enzler H: Air travel for private purposes. An analysis of airport access, income and environmental concern in Switzerland. J Transport Geography. 2017; 61: 1–8. Publisher Full Text\n\nBunn WB, Johnson CE: The business traveler. In: Travel Med. 2019; (pp. 287–293). Elsevier.\n\nCastells M: Globalisation, networking, urbanisation: Reflections on the spatial dynamics of the information age. Urban Studies. 2010; 47(13): 2737–2745. Publisher Full Text\n\nClopper AJ, Baccei EC, Sel TJ: An Evaluation of Zoom and Microsoft Teams Video Conferencing Software with Network Packet Loss and Latency.2020.\n\nCohen SA, Hanna P, Gössling S: The dark side of business travel: A media comments analysis. Transportation Research Part D: Transport and Environment. 2018; 61: 406–419. Publisher Full Text\n\nDeFrank RS, Konopaske R, Ivancevich JM: Executive travel stress: Perils of the road warrior. Acad Management Perspectives. 2000; 14(2): 58–71. Publisher Full Text\n\nDerudder B, Witlox F: International business travel in the global economy. Routledge; 2016.\n\nEcheverria-Arrondo I, Wolfs B: Environmental Perception of Global Business Travel by Swiss Companies in the Zurich Airport Area, 2019-2020. [Data Collection]. Colchester, Essex: UK Data Service; 2021. Publisher Full Text\n\nFaulconbridge JR, Beaverstock JV, Derudder B, et al.: Corporate Ecologies of Business Travel in Professional Service Firms: Working Towards a Research Agenda. Euro Urban Regional Stu. 2009; 16(3): 295–308. Publisher Full Text\n\nFederal Statistical Office: Population [Dataset].2018. Reference Source\n\nAg FZ: 2014 annual report of Flughafen Zürich Ag.2014; Reference Source\n\nAg FZ: 2018 annual report of Flughafen Zürich Ag.2018. Reference Source\n\nGall MD, Gall JP, Borg WR: Collecting research data with questionnaires and interviews. Edu Res: An introduction. 2007; 12(10): 227–261.\n\nGreenhouse Gas Protocol: Scope 2 Guidance.2021; Reference Source\n\nGustafson P: Travel time and working time: What business travelers do when they travel, and why. Time Society. 2012; 21(2): 203–222. Publisher Full Text\n\nGössling S, Hanna P, Higham J, et al.: Can we fly less? Evaluating the ‘necessity’ of air travel. J Air Transport Management. 2019; 81: 101722. Publisher Full Text\n\nHam M, Mrčela D, Horvat M: Insights for measuring environmental awareness. Ekonomski vjesnik: Review of Contemporary Entrepreneurship, Business, and Economic Issues. 2016; 29(1): 159–176.\n\nHarvard Business Review: Managing across distance in today’s economic climate: The value of face-to-face communication. Harvard Business Review Analytic Services Report. 2009; 2009. Reference Source\n\nHepburn C, O’Callaghan B, Stern N, et al.: Will COVID-19 fiscal recovery packages accelerate or retard progress on climate change? Oxford Review of Economic Policy. 2020; 36. Publisher Full Text\n\nHinnen G, Hille SL, Wittmer A: Willingness to Pay for Green Products in Air Travel: Ready for Take-Off? Business Strategy and the Environment. 2017; 26(2): 197–208. Publisher Full Text\n\nInternational Air Transport Association [IATA]: IATA forecast predicts 8.2 billion air travelers in 2037 [Press release].2018, October 24. Reference Source\n\nJulsrud TE, Denstadli JM, Hjorthol RJ: Business Networking, Travel Tiredness, and the Emergent Use of Video Conferences. Int J Sustainable Transportation. 2014; 8(4): 262–280. Publisher Full Text\n\nKagan D, Alpert GF, Fire M: Zooming into Video Conferencing Privacy and Security Threats. arXiv preprint arXiv:2007.01059. 2020.\n\nKolk A: The social responsibility of international business: From ethics and the environment to CSR and sustainable development. J World Business. 2016; 51(1): 23–34. Publisher Full Text\n\nKoyen J: Business meetings: The case for face-to-face. Forbes Insights. 2009.\n\nKuzemko C, Bradshaw M, Bridge G, et al.: Covid-19 and the politics of sustainable energy transitions. Energy Res Social Sci. 2020; p.101685. Publisher Full Text\n\nLo SH, van Breukelen GJP, Peters G-JY, et al.: Pro-environmental travel behavior among office workers: A qualitative study of individual and organizational determinants. Transportation Res Part A: Policy and Practice. 2013; 56, 11–22. Publisher Full Text\n\nMäkelä L, Kinnunen U: International business travelers’ psychological well-being: the role of supportive HR practices. Int J Human Resource Management. 2018; 29(7): 1285–1306. Publisher Full Text\n\nMaxim M, Zander K, Patuelli R: Green tax reform and employment double dividend in European and non-European Countries: a meta-regression assessment. Int J Energy Economics Policy. 2019: 342–355. Publisher Full Text\n\nMueller JL: Drinking the kool-aid: the IMF and global hegemony. Middle East Critique. 2010; 19(2): 93–114.\n\nMugenda OM, Mugenda AG: Res Methods Dictionary. Nairobi, Kenya: Applied Research & Training Services; 2012.\n\nOertig M, Buergi T: The challenges of managing cross-cultural virtual project teams. Team Performance Management: An Int J. 2006; 12(1/2): 23–30. Publisher Full Text\n\nPinzone M, Guerci M, Lettieri E, et al.: Effects of ‘green’ training on pro-environmental behaviors and job satisfaction: evidence from the Italian healthcare sector. J Cleaner Production. 2019; 226: 221–232. Publisher Full Text\n\nPoom A, Orru K, Ahas R: The carbon footprint of business travel in the knowledge-intensive service sector. Transportation Res Part D: Transport Environ. 2017; 50: 292–304. Publisher Full Text\n\nRela IZ, Awang AH, Ramli Z, et al.: Effects of environmental corporate social responsibility on environmental well-being perception and the mediation role of community resilience. Corporate Soc Responsibility Environmental Management. 2020. Publisher Full Text\n\nTani M: On the Motivations of Business Travels: Evidence from an Australian Survey. Asian Pacific Migration J. 2005; 14(4). Publisher Full Text\n\nWeil T, Murugesan S: IT Risk and Resilience—Cybersecurity Response to COVID-19. IEEE Computer Architecture Letters. 2020; 22(03), pp.4–10. - But it is quite technical.\n\nWelch DE, Welch LS, Worm V: The international business traveler: a neglected but strategic human resource. Int J Human Resource Management. 2007; 18(2): 173–183. Publisher Full Text\n\nWestman M, Etzion D: The impact of short overseas business trips on job stress and burnout.2002. Reference Source\n\nWright RW, Ricks DA: Trends in International Business Research: Twenty-Five Years Later. J Int Business Studies. 1994; 25(4): 687–701. Publisher Full Text\n\nYue CA, Men LR, Ferguson MA: Examining the Effects of Internal Communication and Emotional Culture on Employees’ Organizational Identification. Int J Business Communication. 2020; 2329488420914066. Publisher Full Text\n\nAirport Z: Facts and figures. 2018: 2018. Reference Source"
}
|
[
{
"id": "94451",
"date": "28 Sep 2021",
"name": "Stefan Baumeister",
"expertise": [
"Reviewer Expertise Climate Change Mitigation",
"Sustainable Consumption",
"Corporate Responsibility",
"Transportation Sector",
"Environmental Labels",
"Aviation Industry",
"Electric Aviation",
"Carbon Footprint Analysis",
"Modal Shift",
"De-Growth",
"Sustainable Tourism",
"Climate Change Adaptation"
],
"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 research paper addresses an interesting and timely topic as it studies the environmental perceptions of global business travelers in Switzerland. While I very much enjoyed reading the paper and found the findings it provides interesting and to some extent also novel, I must also say that I identified some major short-comings that would need to be addressed before I could recommend indexing this paper.\nA major problem I see is with the sample size which is only 104 survey participants. As the authors pointed out correctly, for this population size the minimum sample size should be 384 participants. As it is common that not all invited potential participants will respond to a survey, I am wondering why you only sent out invitations to 400 individuals when you acknowledge a required minimum of 384 participants? Nevertheless, given the fact that you conduct a mixed-methods approach with in-depth interviews, the low amount of survey participants could be accepted. However, you would need to clarify in your paper that such a small amount of participants significantly reduces the validity of your study and the conclusions drawn from the sample. In addition to that, not collecting demographic data is another major short-coming of this study. This cannot be made up by assuming the demographics. Such information should therefore not be presented in the paper as it could misguide the reader.\nAnother major issue with this study is the lack of literature to back up the hypotheses presented. Although the authors mentioned in the introduction that hypotheses were developed based on secondary literature, these sources were not presented sufficiently in the paper. I would therefore encourage the authors to add a well-developed literature section to the paper that justifies the presented hypotheses clearly, backing them with existing literature. Just listing the hypotheses at the beginning of the methods section isn’t sufficient enough.\nIn the results section I don’t see any need to present/repeat the survey questions in such great detail but to rather focus on the actual results. Also focus should only be placed on the most relevant findings. Please revise the results section accordingly.\nFinally, I find that the discussion section is focusing mainly on the findings of the case study but more or less neglects the rich findings from the survey. As this is a mix-methods approach, I would like to see more discussion on the findings of both studies instead of only the ones from the interviews.\nLast but not least I see in these findings also great potential for policy recommendations the authors could make based on the study. Therefore, I would encourage the authors to add a section on policy recommendations to the discussion section at the end of the section.\nIn addition to these major concerns I have some further detailed comments for the author to considered during the revision:\nIn the results section of the abstract, I would recommend to leave out the exact results from the hypotheses testing as this is not common practice and also difficult for the reader to assess unless he/she is familiar with the hypotheses. The abstract should be a alone-standing document that a reader can fully assess without the need to read the paper as such. Instead of the precise results of the hypotheses testing I would recommend the authors to focus on the actual results the hypotheses tests provided.\nIn the introduction, please rephrase the first sentence as it is confusing. What do you mean with “coronavirus disease 2019 pandemic”? Further in the first sentence of the third paragraph you cite two sources but one as an in-text citation and the other at the end of the sentence. Is there a particular reason for that as this is not common practice?\nIt is very nice to see that the idea for this study was based on a critical assessment of travel behavior by the author himself. However, perhaps you could still use a more general and passive voice when presenting those arguments as the study otherwise appears more like a narrative which it is apparently not.\nIn the background literature section, you cite in the first sentence the Harvard Business Review (2009). Instead of the journal name could you please provide the name of the author of this publication? In the same paragraph you also use the author Tani’s first name initial which should be avoided in in-text citations.\nIn the objectives section I would leave out the word “simple” when presenting the research questions as these are by far not simple questions you are attempting to answer with this study.\nSome aspects in the methods section are in my opinion described in too great detail and not necessarily relevant for the reader to understand how the study was conducted or for researchers to replicate the study, among those were:\nThe questionnaire testing process is described in too much detail\n\nAlternative software that could have been used to conduct the survey\n\nThe name of the person conducting the interviews and the training he received for that\n\nDetails on the use of NDA and its storage\n\nInformation on ethical approval\nInstead, the data analysis could have been described in slightly greater detail.\nThe first two paragraphs of the discussion section are more or less repeating the background and purpose of the study instead of really discussing the actual results. Perhaps those paragraphs could be shortened or left out entirely?\nThe results of the interviews of value for a discussion of broader corporate culture presented in the discussion section are really interesting. Nevertheless, these are results and should not be presented in the discussion section but rather in the results section. Also, instead of presenting these findings in bullet points you might consider presenting them in a table or graph which might be more appealing to the reader.\nFurther comments and an annotated version of the manuscript from my co-reviewers can be found here: Sami El Geneidy, Maija Lähteenkorva.\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Partly\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nNo\n\nAre all the source data underlying the results available to ensure full reproducibility? Partly\n\nAre the conclusions drawn adequately supported by the results? No",
"responses": [
{
"c_id": "8150",
"date": "05 May 2022",
"name": "Ignacio Echeverria Arrondo",
"role": "Author Response",
"response": "Dear reviewer, Thank you for your detailed feedback and please excuse the delay responding, as I was waiting for more reviews. Methodology – as for the sample size, the minimum of 400 respondents (above 384) was requested for the quantitative section of the dissertation from which this article has been extracted, to be accepted (Mugenda and Mugenda 2012). The number of valid survey participants was 104. A comment has been added in the results section; ‘while this sample was sufficient for this mixed-methods study, it is below the 384 required for the quantitative data to be taken as statistically representative of the population being studied. Future researchers may want to replicate this study to a larger sample, and also to take account of the advice in van Mol (2015) and provide repeated digital reminders for potential participants. To prevent lengthening the quantitative survey, which might have further impacted the response rate, demographic data was not collected’. Methodology – the demographic estimated data has been removed as suggested. Background literature – more sources have been added to back up the hypotheses. Results – survey questions have been removed as suggested. Abstract – the results section has been amended although the precise results are still included. In the conclusion ‘research for this article was undertaken prior to COVID-19, so future research will be necessary to investigate the new context’ has been added. Introduction – ‘corona disease 2019 pandemic’ has been rephrased. Also, in-text citation has been amended. Literature background – Tani’s first name initial has been removed, as suggested. In the objective section, the word ‘simple’ has been removed, as suggested. In the opinion of the main author, the questionnaire tests, the NDA and ethical approval are important. Perhaps the name of the interviewer and alternative software could be removed as suggested. The results of the interviews have been moved from the discussion to the results section. All other comments have been partially implemented where and if possible. In the hope that these edits will work to your satisfaction, I would greatly appreciate if you and/or your co-reviewers could review the article and its rating, once the new version has been published in the next couple of days. Kind regards Ignacio Echeverria Arrondo"
}
]
},
{
"id": "119974",
"date": "16 Feb 2022",
"name": "Mònica Guillen Royo",
"expertise": [
"Reviewer Expertise Sustainable consumption",
"mixed methods research",
"wellbeing studies",
"economics of happiness",
"human scale 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 study uses mixed methods to study the relationship between job-related flying practices in companies around the Zurich geographical area and employees perceptions and attitudes towards flying. The paper is well-structured, and the topic is interesting and timely. Nevertheless, lack of clarity concerning the theoretical and methodological underpinnings of the study deem it unsuitable for indexing in its present form.\nGeneral considerations\nIn general, the goal of the study is not clearly presented, and the hypothesis not explicitly grounded on a thorough literature review on the topic. The mixed-methods approach is very suitable for this type of study, but its advantages have not been explicitly discussed and it is unclear for the reader what it is that the qualitative study has added to the results of the descriptive quantitative analysis. The quantitative analysis focusses on the description of variables and a correlational study, but since the general research question is not described, the extent to which the analysis contributes to a greater understanding of corporate travel is unclear.\nDetailed comments\n\nThe abstract should include the main goal of the study (in the background subsection) and results should not refer directly to the hypotheses as the reader does not know what these are at the point of reading the abstract. References to the COVID-19 pandemic might be done in the conclusion but not in the background section as this study was not carried out against the backdrop of the pandemic.\nThe introduction would benefit from including an explicit reference to the goal of the study. It would also be advisable to link the questions that the researcher presents as inspiring the study to a problem in society (health or wellbeing related impacts of flying), the environment (ecological impact of flying) or a gap in the literature that the study wants to fill. Additionally, it is common in academic articles to use the last paragraph of the introduction to describe the sections of the paper and their contents.\nConcerning the background literature section, this should be sufficient to justify the hypotheses presented in the Methods section. At present, it lacks enough depth. The literature on business travel is very extensive. Employees’ values and attitudes have been investigated both drawing on quantitative and qualitative methods and should feature in this section. This is also the case concerning policies to reduce air travel and their lack of effectiveness due to the many barriers and taboos concerning mobility and flying in contemporary societies. The Journal of Air Transport Management, the Journal of Transport Geography and Transportation Research Part D publish many studies that the authors might want to include in this section. Particularly relevant are the works by Stefan Gösling, Scott Cohen and James Higham. Additionally, Sahakian et al. (2021)1 have recently published an article on air travel in (Genève) Switzerland based on document analysis and the analysis of workshop data.\nIn the methods section, some details about ethics clearance and piloting might not be necessary, whilst information about the strategy followed to analyze qualitative data is missing. It is also unclear the bias that the authors would create if they had interviewed respondents themselves. Additionally, it is important to clarify how multistage sampling was applied, if ‘clusters were formed of companies with which the researcher had a pre-existing relationship’, what type of companies were left out? What was the structure of the clusters and how could this sampling strategy be presented as having a random component in it? Moreover, how were the answers to the ‘global business travel’ questions coded, or alternatively were they open questions? Table 1, would be better presented as a descriptive statistics table, where variables are presented in terms of the question, the response scale and measures of central tendency and dispersion.\nWhen presenting the results, it would be useful to uniformize the way the data is presented, using the same type of figures or tables across subsections. In addition, when scales are formed (by adding up the scores of more than two variables) it would be advisable to present the value of Cronbach’s alpha, which would indicate whether there is strong or weak internal consistency of the construct. Furthermore, there seems to be a potential source of confusion concerning the section on business travel and environmental awareness, as it is difficult for the reader to discern why a measure for ‘social responsibility’ is created but later, findings are presented in terms of individual vs. corporate awareness.\nAnother concern in this section is related to one of my previous general comments, why is the qualitative information important? How does it help in understanding the relationship addressed in this section? The biggest challenge here is that the goal of the paper is unknown, so the reader cannot easily follow what the article is trying to achieve. If the goal was to understand how environmental perceptions affect business travel, then one would expect regression analysis to be used to explain the determinants of business travel frequency for example, but this is not done here probably because of the small sample size and the lack of availability of socio-demographic variables.\nFinally, Ha (5) is difficult to investigate drawing on a variable that captures results from a hypothetical question. Studying this hypothesis would require having data about the extent to which corporate social responsibility policies are in place in each company, which does not seem to be the case in the survey of this study.\nOne expects the discussion section to go back to the literature described in the second section to discuss the results of the study. Listing the topics raised by interviewees does not appear to add much to the discussion and might be removed. The paragraph on videoconferencing refers strongly to the COVID-19 pandemic, and although this might be a topic for further research cannot be used here to draw conclusions on policy as the study was carried out before the COVID-19 outbreak. An important finding of the study is the one concerning corporate communication. Probably the article and the analysis would increase their relevance if they had been articulated around this very important topic.\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?\nNo\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? No",
"responses": [
{
"c_id": "8151",
"date": "05 May 2022",
"name": "Ignacio Echeverria Arrondo",
"role": "Author Response",
"response": "Dear reviewer, Thank you for your detailed feedback, and please excuse the delay in responding. Abstract – in the new version, the background section was rephrased to include the goal, and the results are not referring directly to the hypotheses, as suggested. Introduction – an explicit reference to the goal of the study was added as well as an outline of the article’s structure, as suggested. Additions were made to position the article more clearly as contributing to the evidence base around CSR, international business travel, and employee wellbeing. Background literature – references were added, after revisiting notes from the doctoral thesis from which this article emerged and reviewing more recent work on employee responses to air travel, as well as per your suggestions. Several edits were made to ensure it is clear to the reader that the article provides evidence to demonstrate, largely in line with previous literature, that awareness is insufficient; action must be taken. Some suggested actions which emerged from the interviews and research were added in a new paragraph in the conclusion. Methodology – in the opinion of the main author, piloting, NDA and ethical clearance are important in this case. Perhaps the name of the interviewer and alternative software could be removed. Results – this section also contains several edits. Clearer figure captions. A reduction in the repetition of the survey questions. An explanation of how the combined variables were created. A few quotes from the interviews were added, and the broader “inputs of value for a discussion of broader corporate culture” from the interviews, which was in the conclusion in the previous version, have been brought up in the results section. Concerning the H(5) justification for the usage of hypothetical questions within the set, it was explained in a new paragraph as part of the discussion of the quantitative methodology, with reference to appropriate literature. References and evidence have been added. All other comments have been partially implemented where possible. In the hope that these edits will work to your satisfaction, I would greatly appreciate if you could review the article and its rating, once the new version has been published in the next couple of days. Kind regards Ignacio Echeverria Arrondo"
}
]
}
] | 1
|
https://f1000research.com/articles/10-925
|
https://f1000research.com/articles/11-1570/v1
|
23 Dec 22
|
{
"type": "Method Article",
"title": "Turning any bed into an intensive care unit with the Internet of things and artificial intelligence technology. Presenting the enhanced mechanical ventilator",
"authors": [
"Leidy Lorena Pulido Morales",
"Juan Sebastian Buitrago Romero",
"Ismael A. Ardila Sanchez",
"Fernando Yepes-Calderon",
"Leidy Lorena Pulido Morales",
"Juan Sebastian Buitrago Romero",
"Ismael A. Ardila Sanchez"
],
"abstract": "The recent Coronavirus disease 2019 (COVID-19) pandemic displayed weaknesses in the healthcare infrastructures worldwide and exposed a lack of specialized personnel to cover the demands of a massive calamity. We have developed a portable ventilator that uses real-time vitals read from the patient to estimate -- through artificial intelligence -- the optimal operation point. The ventilator has redundant telecommunication capabilities; therefore, the remote assistance model can protect specialists and relatives from highly contagious agents. Additionally, we have designed a system that automatically publishes information in a proprietary cloud centralizer to keep physicians and relatives informed. The system was tested in a residential last-mile connection, and transaction times below the second were registered. The timing scheme allows us to operate up to 200 devices concurrently on these lowest-specification transmission control protocol/internet protocol (TCP/IP) services, promptly transmitting data for online processing and reporting. The ventilator is a proof of concept of automation that has behavioral and cognitive inputs to cheaply, yet reliably, extend the installed capacity of the healthcare systems and multiply the response of the skilled medical personnel to cover high-demanding scenarios and improve service quality.",
"keywords": [
"Technology in healthcare",
"Artificial intelligence in medicine",
"Covid 19 mitigation",
"Intense care units everywhere",
"mechanical ventilators",
"AWS implementations",
"Evalu@ implementations",
"Artificial Neuronal Networks in medicine"
],
"content": "Introduction\n\nCurrently, the world population is passing through one of the most significant viral outbreaks in the modern era. The infectious agent, severe acute respiratory syndrome Coronavirus 2 (SARS-CoV-2), spreads fast and uses humans as vectors, a threat to our kind never seen before.1\n\nSARS-CoV-2 produces Coronavirus disease 2019 (COVID-19), a health condition characterized by respiratory infections. The level of affliction ranges from simple cold symptoms to severe illnesses that lead to general failure and death.2\n\nThe Centers for Disease Control and Prevention (CDC) reported 624,088,072 confirmed cases and 6,552,725 deaths caused by COVID-19 worldwide by October 4th, 2022.3\n\nApproximately 80% of the world’s positive cases for COVID-19 recover from the disease without medical treatment.4 The other 20%, including young individuals without pre-existing conditions, the elderly, and people with chronic illnesses, might present a severe affliction that could compromise the immune system and organs such as the lungs, heart, kidneys, liver, and brain.5,6\n\nThe rapid spreadability of SARS-CoV-2 made contamination intensify over time, with exponential growth trending.7 No country or health system in the world proved to have the infrastructure, resources, and response capacity to attend to the demand for care during contagion peaks.8 With the intense care units (ICU) filled and medical personnel exhausted, any bed turned into a potential care unit; however, ventilators and specialized professionals are more challenging to find.9 The scarcity of ventilators and specialized personnel repeated with every peak suffered worldwide.\n\nWhen developing this work, South Korea, Vietnam, and Germany passed through a new peak of contamination with more than 200,000 new cases per day and more than 70 causalities in the same period, even when the cited countries reported vaccination done to 100% of the population.10\n\nThis research reviewed mechanical setups, programmable electronics, embedded telecommunications, application programming interface (API) services, artificial intelligence (AI) implementations, and human respiratory variables to build a low-cost and highly reliable mechanical ventilator (MV). After reading the sensors, the device integrates a neuronal network capable of making control decisions in a closed loop that modifies the range and operating frequency of the device.\n\nAdditionally, the device reports the readings and control actions wirelessly to a data centralizer that can display the variables in real-time to any screen – including cell phones – lessening the need for dedicated and specialized personnel working in close contact with the infectious patients.\n\n\nMethods\n\nThis section describes the construction of a non-invasive MV controlled by a feedback artificial neural network (ANN). The developed MV reads the patient’s heart rate, oxygenation percentage, and respiratory pressure; and dynamically defines the working frequency of the ventilator and, consequently, the amount of oxygen to deliver.\n\nSince low-cost sensors are involved in the MV’s operation, we trained an ANN with preconceived ‘true’ data to correct the outputs; therefore, the ANN makes the right decisions with an acceptable low rate of uncertainty regarding oxygen supply. The ventilator and the ANN are connected wirelessly within an AD-HOC network in a star topology that facilitates data transferring between a theoretically unlimited number of nodes. In addition, the effort to link new devices is low compared with firmware-based protocols such as the message queuing telemetry transport (MQTT).11 Since the system uses the transmission control protocol/internet protocol (TCP/IP) protocol, the number of available nodes depends on the IP address class, the most common being the C class with 254 available nodes (assuming no subnetting). Recall that TCP/IP uses 32 bits logic addresses classified in A, B, C, D and E depending on how many octets are employed for allocating hosts and their purpose. Class C addresses are the ones with less hosting allocation capabilities and give Internet access to household residences. Since C class addresses reserve one octet for hosts, the current residential infrastructure can allocate nd = 254 devices (nd = 28 − 2).\n\nA programmable device like BeagleBone Black (BBB) is the local brain of the system, synchronizing the sensors (inputs) and the actuator (output). This local brain interchanges information with the Amazon Web Service (AWS)[1] that allocates the ANN and returns operating configurations. The BBB and the AWS use an ESP32 chip as a data gateway with a universal asynchronous receiver transmitter (UART) to connect the BBB and TCP/IP on the side of the ANN. Using a homemade API, the gateway also pushes the estimated operation points, and the patient’s vitals to the Evalu@ service.12 Since medical applications do not admit information lost (insufficient information or delayed data due to packets lost will affect the decision stage and eventually the patient’s health), we sacrificed speed and preferred reliability by choosing TCP instead of the user datagram protocol (UDP). Recall that UDP is a faster protocol with no recovery mechanisms in case information packets are corrupted or lost.13 The firmware is written in C, ensuring faster operation than other programming platforms.14 See a general diagram of the AI-based ventilator in Figure 1.\n\n\n\nWe designed the core structure of the ventilator with a plastic resin to provide resistance to the mobile parts while making the device portable and durable. A servomotor connected to the scissors-like levers activates the degree of freedom associated with the scissors’ opening angle that ultimately presses a flexible container. A predefined rotation setup exerted by the servomotor pushes the needed air to the pumping container, thus, to the patient. The system controls the air’s volume and delivery frequency with this arrangement. The ventilator has a buzzer, lighting, and a 16x2 matrix screen to account for in-site alarms and state displaying. See the ventilator’s physical appearance in Figure 2.\n\nThe MPX2010 differential pressure sensor, developed by Freescale semiconductors ®, has a linear transfer function f = 2.5/1[mV/KPa], and an output voltage span of [0-25 mV].15 The sensor yields a pressure value (Pi) by subtracting the input (P1i) – connected indirectly through the pumping system to the patient’s airways – from the value exerted in the vacuum input (P2i). An adjusting factor Af = 0.17 affects every Pi in the P array to translate the readings from KPa to MBAR units (See Equation 1).\n\nThe patient’s pulmonary pressure is calculated from the maximum values in the P array.\n\nRegarding the heart rate (HR) and oxygen saturation, the MAX30100 chip developed by Maxim Integrated ®, reads both variables. It uses a visible red light (660 nm) and a receptor to capture intensity reflections after hemoglobin changes due to heart pulsation.16 As for the oxygen saturation, the chip implements a second source of light working at the near-infrared spectrum (880 nm). The module determines the absorption spectrum of oxygenated and deoxygenated hemoglobin with the two light sources that produce interchange reflection peaks at the respective wavelengths. The MAX30100 digitizes the reflections of both light beams with a resolution of V cc/216 and returns the sensed variables in human-readable format through the Inter-Integrated Circuit (I2C) interface.\n\nWe designed a six-layered ANN (See Figure 3). The input layer has three nodes to receive the heart rate, oxygenation percentage, and respiratory pressure. The output layer consists of one node, which provides the dynamic operation setup for the servomotor and ultimately controls the oxygen yield to the patient. The training data consisting of 1000 formulations with the three used features, and the supervising variable is available online here.17 The training data was gathered from ICU records performed by Instituto de Genetica (Genesis S.A.S). The data is fully anonymized and respect the health insurance portability and accountability act (HIPAA)18 directives.\n\nThe initial layer receives the three vitals of the patient, and the output node returns the operation point that controls the amount of delivered oxygen.\n\nThe ANN uses two activation functions, namely Sigmoid and Identity, consequently, the system has a dynamic input/output ratio. The activation functions simulate the evoked potential that controls the release of neurotransmitters within the neurons in alive subjects. In the ANN context, the identity function is a mathematical activation model governed by the expression f(x) = x; therefore, it transfers the input to the output in a range (∞,∞). The sigmoid or logistic activation function, also known as a binary transfer, is governed by the expression fx=11+e−x and its range is (0, 1). The training process consists in adjusting the nodes’ outputs, so the combined work of the network yields numbers close to the supervising values. Backpropagation with a controlled gradient descent was implemented to modify the weights in the internal ANN layers while searching for optimization. The controlled gradient descent is accomplished using the Levenberg-Marquard strategy (trainlm)19 that converges fast to optimization even when a wrong initial optimization guess is selected and provides mechanisms to avoid oscillations around the optimal value. The trainlm, is a distance minimization algorithm that receives an initial guess from the user and interactively updates a variable β. The method is fully implemented in python and is available in https://github.com/jjhartmann/Levenberg-Marquardt-Algorithm\n\nSharing and processing the sensor data between the ESP32 and the AWS was accomplished through the lambda specification.20 We start by provisioning a Virtual Private Cloud (VPC)21 with the necessary utilities to connect and display the reports of the services to be consumed. Within the VPC architecture, one lambda triggers the microservice to establish the connection and feed the ANN with the three patient vitals. A second lambda triggers the microservice to obtain the ventilator setup yield by the ANN, which is delivered to the device’s hardware.\n\nPatients’ data and actions taken by the ANN are sent to the Evalu@ service for storing, querying, analysis and reporting. This centralizer allows custom reports creation and online analysis through intuitive Excel templates, providing the flexibility to produce, in real-time, material to healthcare providers and relatives of the patients as well. A copy of the control values yielded by the ANN is also stored in the Dynamo DB service22 of the AWS.\n\nAfter establishing the ANN architecture, we ran a standard validation process by comparing the supervisor factors with the outputs yielded by the ANN. For the validation, we performed a three-folded exercise using 30% on the training data referred to in section Artificial neural network construction. We randomly selected the data for each folding. We obtained a 95.79% accuracy during these testing sessions using a Sigmoid activation function. The accuracy index decreased to 94.01% when using the Rectified Linear Unit (ReLU),23 and the best performance regarding accuracy appeared when using a combination of Sigmoid and Identity functions among the ANN layers.\n\nThe Evalu@ service (here) provides an intuitive mechanism to configure any tracking/monitoring scheme. The platform interprets the entries of three editable Excel files to create containers for the items to be evaluated, the tracking instruments, and the analysis that should be performed with the gathered data. Figure 4 presents the configuration files for the current application and Evalu@’s starting interface once the setup is finished.\n\nPanels A, B, and C show the configuration files used to create the containers, the tracking instruments, and the indexes’ visualization, respectively. Panel D displays the main screen after setup completion.\n\nEvalu@ is needed to present the data appealingly to users other than developers. It can also allocate the services executed now in AWS. However we leave this to further developments. Readers can now reproduce our methods using AWS and the provided code. If requiring the use of Evalu@, code C08 in the Zenodo repository displays the API to do so, and users testing the presented methods can use the Evalu@ service for free.\n\n\nResults\n\nThe Table 1 shows the performance of the python ANN implementation with the selected configuration, using a combination of Sigmoid and Identity functions among the ANN layers.\n\nWe ran data processing tests on the services exposed on the ESP32 (gateway) regarding the AWS and Evalu@ services. The records in Table 2 are the response times for one ventilator.\n\n\n\nWith the records in Table 2, we estimated the bursting capacity of the solution by simulating 200 concurrent users, adding to a total latency of 12 seconds. Recall that Internet connectivity in residences, where this development is intended to work, can allocate a maximum of 254 devices; however, since wifi performs like having the devices connected through a hub, latency can exponentially deteriorate when the technical limit of connections is reached.\n\nThe VPC at AWS presents an interface to visualize the system’s architecture and implemented jobs. Figure 5 shows the AWS API gateway flow map for processing the breathing frequency.\n\nThe AWS produces control values to set the ventilator’s operation. We store the control setups produced by the ANN in the Dynamo DB as shown in Figure 6.\n\n\n\nAlthough AWS can intuitively present the generated information, their displaying schemes are intended for development and debugging. Not to mention the commercial strategy that charges the user after reaching an established quota. Instead, Evalu@ has mechanisms to present the data to non-specialized users. We employ Evalu@ to present physicians and patients’ relatives with real-time information on vitals and control variables (see Figure 7).\n\nThe vitals are presented without alarm levels. The control signal has all the levels activated. It generates mail alarms – in addition to those generated in-site – when the control signal is above or below the established levels.\n\nThe Figure 8 resumes one loop operation of the system and provides step-by-step linking to the code deposited in https://doi.org/10.5281/zenodo.7400986. The system starts by configuring the ports and screen (C01). Then, it will read and process the patient’s vitals (C02-C03). The ventilator sends the vitals to the AWS using the ESP32 and the API (C04). The AWS uses the lambda specification to feed the ANN with the vitals and recovers the breathing-frequency variable (C05), which is saved in the DynamoDB (C06). The ESP consumes the API (C07) to recover the breathing frequency used by the ventilator to update the duty cycle of the Ambu system. The ESP also sends the vitals and control variables to the Evalu@ service using the API presented in C08. The authors encourage using the AWS S3 service, which will be enough to reproduce the results presented in this document. If an interface is needed to show the results to non-technical users, the authors warrant using Evalu@ at no cost for this particular application.\n\nThe rounded symbols refer to code supporting the functionality that has been presented as complementary material to assert reproducibility.\n\n\nDiscussion\n\nDuring the SARS-CoV-2 outbreak and due to the high demand for mechanical respirators, developers responded with prototypes intended for intensive care unit (ICU) use.24,25 Additionally, medical personnel worked extended hours to cover the high demand for specialists and exposed themselves to the viral agent.26 Companies and independent developers rushed to build prototypes and devices to cover the primary necessity at the cost of the high demand. However, the shortage was not only in materials but in personnel.\n\nThe presented design aims to assist patients in any place where one can improvise a bed while accounting for the shortage of specialized personnel by providing the systems with autonomous decision capabilities based on artificial intelligence. We also propose using ad hoc. networks to maintain distance with aerial and rapid transmission agents. The proposed strategy does not exclude the healthcare specialists; instead, it suggests that a cooperative environment employing highly available systems with configurable alarms will enhance the working environment leading to higher productivity while protecting healthcare personnel from aerial pathogens.\n\nThe created ventilator exploits telemedicine and allows health professionals to assist several patients with a glance at the reporting screen provided by Evalu@ with worldwide coverage, authenticated access, and adherence to HIPAA regulations.18\n\nThe developed device can make autonomous decisions in real time. Professionals can attend to a larger group of people by monitoring their vital signs and prioritizing patients with more severe complications. In addition, the device can trigger alarms by value or trend so that care is no longer dedicated but demand-driven. Such a strategy positively impacts relevant aspects like efficiency and operating costs.\n\nThe proposed architecture uses standard telecommunications protocols, such as the TCP over WiFi (wireless fidelity) or GSM (global system for mobile communication) networks, assuring message integrity. Moreover, the SigFox27 protocol can be implemented as we did in a previous research-transferred development that reached commercialization28 to warrant operation in rural zones.\n\nThe implemented design is not rigid; therefore, we can add more sensors, and both the AWS and Evalu@ have the flexibility to afford the increase in processing and workload.\n\nWe are currently working on transferring the whole ANN processing to Evalu@ since the platform belongs to SBP Research and the research team led by author FYC. Evalu@ presents advantages regarding usability without incurring high costs derived from the expected massive use and long-term operation.\n\nThis development complements the devices created as proofs of concept supporting Patent No US20200273551A1.29 The patent claims healthcare can move from a curative/preventive perspective to a predictive scheme where we can anticipate maladies occurrences using AI, which increases survival rates while making more efficient use of healthcare funds. The patent protects the design of an architecture that enables massive data gathering intended for artificial intelligence implementations. How the data is produced – algorithms and feeding devices – is out of the patent’s scope.\n\n\nConclusions\n\nIn the apparent decline of the most recent pandemic, humanity learned – the hard way – several crucial lessons. We know now that pathogens can turn off worldwide activities, kill massively, and use humans as infectious vectors; a combination of factors compromising the existing infrastructure and rendering resources insufficient. The impact of further attacks would depend on how we optimize the resources. The inclusion of technology has the potential to place goods and means everywhere. Additionally, AI enables the reproduction of cognitive skills at a low budget to face difficulties with enhanced capabilities and be more efficient in calmed days. The presented ventilator is a proof of concept to demonstrate the feasibility of distributed healthcare that is assisted by reasonably cheap technology. It also automatically gathers reliable data that progressively empowers AI to derive verdicts and increase the accuracy of automated decisions.\n\n\nEthics and consent\n\nThe patient data presented is taken from an author who volunteered to test the equipment presented. Ethical approval was not sought out for this study as it is considered to be of low risk and is not an intrusive test. There were no drugs or contrast agent administered and the author was awake at all times.",
"appendix": "Data availability\n\nZenodo: Human Respiration Dataset - Training purposes https://doi.org/10.5281/zenodo.7324274. 17\n\nThis project contains the following underlying data:\n\npatientA_30secs.csv holding the records of vitals read on a healthy individual and control setups for a 5-minutes run.\n\ntrainingDS.csv is a 1000 formulations file listing the three features and the supervising factor used during training and validation of the ANN.\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).\n\n\nReferences\n\nCiotti M, Ciccozzi M, Terrinoni A, et al.:The covid-19 pandemic. Crit. Rev. Clin. Lab. Sci. 2020; 57(6): 365–388. Publisher Full Text\n\nPurohit D, Ahirwar AK, Sakarde A, et al.:Covid-19 and lung pathologies. Horm. Mol. Biol. Clin. Invest. 2021; 42(4): 435–443. Publisher Full Text\n\nCenters for Disease Control and Prevention. Covid data tracker.2022. Accessed = 2022-10-04.Reference Source\n\nLedford H:Why do covid death rates seem to be falling? Nature. 2020; 587(7833): 190–192. PubMed Abstract | Publisher Full Text\n\nJamwal S, Gautam A, Elsworth J, et al.:An updated insight into the molecular pathogenesis, secondary complications and potential therapeutics of covid-19 pandemic. Life Sci. 2020; 257: 118105. PubMed Abstract | Publisher Full Text | Free Full Text\n\nGavriatopoulou M, Korompoki E, Fotiou D, et al.:Organ-specific manifestations of covid-19 infection. Clin. Exp. Med. 2020; 20(4): 493–506. PubMed Abstract | Publisher Full Text | Free Full Text\n\nSrivastava N, Preeti Baxi RK, Ratho, et al.:Global trends in epidemiology of coronavirus disease 2019 (covid-19). Coronavirus disease 2019 (COVID-19). Springer;2020; pages 9–21.\n\nSen-Crowe B, Sutherland M, McKenney M, et al.:A closer look into global hospital beds capacity and resource shortages during the covid-19 pandemic. J. Surg. Res. 2021; 260: 56–63. PubMed Abstract | Publisher Full Text | Free Full Text\n\nRuiz-Fernández MD, Ramos-Pichardo JD, Ibáñez-Masero O, et al.:Compassion fatigue, burnout, compassion satisfaction and perceived stress in healthcare professionals during the covid-19 health crisis in spain. J. Clin. Nurs. 2020; 29(21-22): 4321–4330. PubMed Abstract | Publisher Full Text\n\nXavier CR, Oliveira RS, da Fonseca V , et al.:Timing the race of vaccination, new variants, and relaxing restrictions during covid-19 pandemic. J. Comput. Sci. 2022; 61: 101660. PubMed Abstract | Publisher Full Text | Free Full Text\n\nSarierao BS, Prakasarao A:Smart healthcare monitoring system using mqtt protocol. 2018 3rd international conference for convergence in technology (I2CT). 2018; pages 1–5. IEEE.\n\nYepes-Calderon F, Yepes Zuluaga JF, Yepes Calderon GE:Evalu@: an agnostic web-based tool for consistent and constant evaluation used as a data gatherer for artificial intelligence implementations. International Conference on Applied Informatics. Springer;2019; pages 73–84.\n\nAL-Dhief FT, Sabri N, Latiff NMA, et al.:Performance comparison between tcp and udp protocols in different simulation scenarios. International Journal of Engineering & Technology. 2018; 7(4.36): 172–176.\n\nPrechelt L:An empirical comparison of c, c++, java, perl, python, rexx and tcl. IEEE Computer. 2000; 33(10): 23–29. Publisher Full Text\n\nBaum J:Low-pressure sensing with the mpx2010 pressure sensor. Motorola application note AN1551/D.\n\nSuganthi Evangeline C, Lenin A:Human health monitoring using wearable sensor. Sens. Rev. 2018; 39: 364–376. Publisher Full Text\n\nLeydi L:Pulido Morales; Juan S. Buitrago Romero; Ismael A. Ardila Sanchez; Fernando Yepes-Calderon. Human respiration dataset (1.0).2022a. Accessed = 2022-12-05. Publisher Full Text\n\nAnnas GJ:Hipaa regulations: a new era of medical-record privacy?. N. Engl. J. Med. 2003; 348: 1486–1490. PubMed Abstract | Publisher Full Text\n\nMustafidah H, Hartati S, Wardoyo R, et al.:Selection of most appropriate backpropagation training algorithm in data pattern recognition. arXiv preprint arXiv:1409.4727. 2014.\n\nCrespo-Cepeda R, Agapito G, Vazquez-Poletti JL, et al.:Challenges and opportunities of amazon serverless lambda services in bioinformatics. Proceedings of the 10th ACM International Conference on Bioinformatics, Computational Biology and Health Informatics. 2019; pages 663–668.\n\nVaria J, Mathew S, et al.:Overview of amazon web services. Amazon Web Services. 2014; 105.\n\nSivasubramanian S:Amazon dynamodb: a seamlessly scalable non-relational database service. Proceedings of the 2012 ACM SIGMOD International Conference on Management of Data. 2012; pages 729–730.\n\nAgarap AF:Deep learning using rectified linear units (relu). arXiv preprint arXiv:1803.08375. 2018.\n\nIyengar K, Bahl S, Vaishya R, et al.:Challenges and solutions in meeting up the urgent requirement of ventilators for covid-19 patients. Diabetes Metab. Syndr. Clin. Res. Rev. 2020; 14(4): 499–501. PubMed Abstract | Publisher Full Text | Free Full Text\n\nPearce JM:A review of open source ventilators for covid-19 and future pandemics. F1000Res. 2020; 9: 218. PubMed Abstract | Publisher Full Text | Free Full Text\n\nLefèvre H, Stheneur C, Cardin C, et al.:The bulle: Support and prevention of psychological decompensation of health care workers during the trauma of the covid-19 epidemic. J. Pain Symptom Manag. 2021; 61(2): 416–422. PubMed Abstract | Publisher Full Text | Free Full Text\n\nFourtet C, Ponsard B:An introduction to sigfox radio system. LPWAN Technologies for IoT and M2M Applications. Elsevier;2020; pages 103–118.\n\nYepes-Calderon F, Quiceno AFG, Orozco JFC, et al.:The bio-i capsule. Preventing contagion of aerial pathogens with real-time reporting in Evalu@. ICAI. 2020.\n\nYepes-Calderon F, McComb Gordon J.:Enabling the centralization of medical derived data for artificial intelligence implementations.2020. Accessed = 2022-10-10.Reference Source\n\nPulido Morales LL, Buitrago Romero JS, Ardila Sanchez IA, et al.:Ai based ventilator code (1.0).2022b. Accessed = 2022-12-05. Publisher Full Text\n\n\nFootnotes\n\n1 S3 specification - free service until 5G/Year with AI plugins can be found here"
}
|
[
{
"id": "158688",
"date": "06 Jan 2023",
"name": "Christian Grévisse",
"expertise": [
"Reviewer Expertise Computer science",
"e-learning applied to 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 authors describe a portable ventilator to enable remote monitoring and handling of patients in contexts of contagious agents. The motivation behind this is to cover both lack in specialists and material in high-demand settings such as the COVID-19 pandemic. The device comprises sensors to measure a patient's heart rate, oxygen saturation and pulmonary pressure. These vital signs are given to an artificial neural network (ANN) which estimates the breathing frequency. This parameter then controls mechanical arms around an Ambu bag.\nAI:\nThe authors do not explain why an ANN has been chosen, compared to other machine learning methods. It would be helpful to justify this choice or even compare it.\n\nThere is insufficient information on the training data, i.e., no indication about age, gender or pathologies. This also raises the question for which population (age, gender, pathologies) this ventilator can be used.\nDevices:\nAre the sensors proven to provide accurate data (e.g., approval through the FDA), or were their data compared to clinical instruments?\n\nThe paper provides little information on the home-made ventilator itself. The authors should provide reconstruction plans and overall indicate fees and sustainability aspects (e.g., materials, energy consumption).\nNetworking:\nWhy is an ad-hoc Wifi network used? The explanation about maintaining distance with aerial and rapid transmission agents (second paragraph of the \"Discussion\" section) is not very sound. In any case, a connection to the Internet is required to establish connection to the two remote services (AWS and Evalu@). Please further explain the downsides of an infrastructure Wifi.\nRemote services:\nAs the data from the ANN is crucial for the operation of the ventilator, should the ANN not rather be run on a local device to avoid negative effects of network latency, congestion or unavailability?\nPrivacy:\nDid the authors consider privacy-related issues concerning the fact that patient data would be transferred to two remote services (AWS and Evalu@)?\n\nHow (and by whom) is access to patient data on Evalu@ granted or revoked? What security measures are in place to protect this sensitive data?\nTesting and validation:\nThe paper mentions a volunteer, but there are no indications about their age, gender and health condition. The section \"Evalu@ records on healthy volunteer\" does not provide any information on the volunteer either, albeit mentioning them in the title.\n\nHas the device been tested on a high-fidelity manikin simulating different pathologies?\n\nHas the approach been validated by an emergency medicine specialist?\nGeneral comments:\nIn the paragraph before the \"Discussion\" section, the authors mention about updating the duty cycle of the Ambu system. For audiences not familiar with emergency medicine, it would be beneficial to further explain this.\n\nIn the first paragraph of the \"Discussion\" section, the authors briefly mention the prototypes developed during the high demand for respirators at the outbreak of the COVID-19 pandemic, yet fail to provide further details on similar approaches reported in the literature. It would be beneficial to compare other approaches to the setting proposed in this paper.\n\nFigures 5+6: Maybe redo these screenshots with English locale.\n\nFigure 8: Typo in the lower-left rectangle (\"Control Ventitlator\").\n\nFormatting issue in reference 17\n\nConclusion: Do the authors have any plans for future work in this context?\nCode:\nThere is no code for controlling the ventilator after receiving the ventilation frequency from AWS.\n\nThere is a syntax error in C05, second to last line.\nOverall, the method proposed in this paper is promising, enabling a cooperative environment helpful for telemedicine settings during high-demand periods where lack of personnel can happen. However, during the beginning of the COVID-19 pandemic, the world faced a huge supply chain issue regarding silicon. As the proposed ventilator requires quite some electronic equipment, the lack of material could hamper the approach. What solutions do the authors see concerning this issue?\nThe article should be further reviewed by an expert in AI methods as well as an emergency medicine specialist.\n\nIs the rationale for developing the new method (or application) clearly explained? Yes\n\nIs the description of the method technically sound? Yes\n\nAre sufficient details provided to allow replication of the method development and its use by others? Partly\n\nIf any results are presented, are all the source data underlying the results available to ensure full reproducibility? Partly\n\nAre the conclusions about the method and its performance adequately supported by the findings presented in the article? Partly",
"responses": []
},
{
"id": "158687",
"date": "25 Jan 2023",
"name": "Andres Felipe Montaño",
"expertise": [
"Reviewer Expertise Electronic system",
"Robotics",
"software design"
],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis paper describes the hardware and software architectures of a portable mechanical ventilator (including data management and the user interface) that uses artificial intelligence to estimate its optimal operation point. It is motivated by the need to provide high quality service in high-demand scenarios, such as the recent COVID-19 outbreak.\nIt measures the patient's heart rate, oxygen saturation, and pulmonary pressure as inputs. The sensors used to obtain this information are described. An artificial neural network (ANN) uses this information to estimate the breathing frequency. Using this parameter, mechanical arms are controlled around an Ambu bag to provide oxygen to the patient.\nIn general, the motivation and objectives are relevant, and the paper is well written and easy to read. The introduction is very clear and explains very well the motivation of the work. This seems a mature work, result of integration of the experience of the author in related fields (hardware and software design).\nSome comments are as follows.\nDescription of the system is clear, but the authors could provide more detailed information of the mechanical parts of the system.\nTo enrich the content of the article, a discussion of the decisions in designing the neural network and the motivation for choosing an ANN could be added. Information provided in Figure 3 is not relevant for understanding the behavior or design discussion of the system. The ANN appears to be a critical component of the system and the decision of implementing it in an external service such as AWS should be better supported. At least explain why it is not implemented locally, leaving only the database and the display information to the external services provided by the internet (through AWS and Evalu@). In my opinion, an AI expert should review the article.\nThere should be a more detailed comparison with previous/similar works (including in the references but not remarked/compared with the proposed system).\nPlease, review/evaluate the content of Figures 5 and 6, if they are considered relevant update labels from Spanish to English, but, for example, part B of figure 6 does not seem to add information to the paper.\n\nIs the rationale for developing the new method (or application) clearly explained? Yes\n\nIs the description of the method technically sound? Yes\n\nAre sufficient details provided to allow replication of the method development and its use by others? Yes\n\nIf any results are presented, are all the source data underlying the results available to ensure full reproducibility? Partly\n\nAre the conclusions about the method and its performance adequately supported by the findings presented in the article? Partly",
"responses": []
},
{
"id": "158686",
"date": "13 Feb 2023",
"name": "Maria Florencia Pollo-Cattaneo",
"expertise": [
"Reviewer Expertise Information technology",
"artificial intelligence",
"machine learning",
"digital transformation"
],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 proposes to provide a respiratory assistance service (portable mechanical ventilator) using technologies provided by artificial intelligence to optimize its operation. It includes the hardware and software architecture used, data management and interfaces.\nThe problem is approached from the pandemic produced by COVID-19 and the hospital requirements in the face of the worldwide health crisis. The topic is relevant and of interest. The motivation is genuine and seeks to improve people's quality of life.\nThe objective and methodology used is correct and consistent with the approach taken. Some sources of similar or previous works on the subject are not identified.\nThe mechanical processes used and the ANNs used are described.\n\nThe results are promising and it is perceived that it is an advanced work.\nThe conclusions are relevant. Future lines of work are still to be identified and if possible a medical evaluation by a professional specialist in the area should be available.\n\nThe following suggestions are made:\nTo revise the texts of some figures and their language.\n\nIt is not clear the selection criteria of the chosen neural network topology. Nor why this technology is used.\n\nA description of why AWS is used could be complemented.\n\nTo revise the texts of some figures and their language.\n\nIt is recommended to have a technical report describing the mechanics and construction of the system.\n\nTo consider in future lines scalability issues and possible conflicts (of connectivity and own of the development of the mechanics of the ventilator).\n\nIs the rationale for developing the new method (or application) clearly explained? Yes\n\nIs the description of the method technically sound? Yes\n\nAre sufficient details provided to allow replication of the method development and its use by others? Partly\n\nIf any results are presented, are all the source data underlying the results available to ensure full reproducibility? Partly\n\nAre the conclusions about the method and its performance adequately supported by the findings presented in the article? Yes",
"responses": []
}
] | 1
|
https://f1000research.com/articles/11-1570
|
https://f1000research.com/articles/11-493/v1
|
04 May 22
|
{
"type": "Software Tool Article",
"title": "SL-Cloud: A Cloud-based resource to support synthetic lethal interaction discovery",
"authors": [
"Bahar Tercan",
"Guangrong Qin",
"Taek-Kyun Kim",
"Boris Aguilar",
"John Phan",
"William Longabaugh",
"David Pot",
"Christopher J. Kemp",
"Nyasha Chambwe",
"Ilya Shmulevich",
"Bahar Tercan",
"Guangrong Qin",
"Taek-Kyun Kim",
"Boris Aguilar",
"John Phan",
"William Longabaugh",
"David Pot",
"Christopher J. Kemp"
],
"abstract": "Synthetic lethal interactions (SLIs), genetic interactions in which the simultaneous inactivation of two genes leads to a lethal phenotype, are promising targets for therapeutic intervention in cancer, as exemplified by the recent success of PARP inhibitors in treating BRCA1/2-deficient tumors. We present SL-Cloud, a new component of the Institute for Systems Biology Cancer Gateway in the Cloud (ISB-CGC), that provides an integrated framework of cloud-hosted data resources and curated workflows to enable facile prediction of SLIs. This resource addresses two main challenges related to SLI inference: the need to wrangle and preprocess large multi-omic datasets and the availability of multiple comparable prediction approaches. SL-Cloud enables customizable computational inference of SLIs and testing of prediction approaches across multiple datasets. We anticipate that cancer researchers will find utility in this tool for discovery of SLIs to support further investigation into potential drug targets for anticancer therapies.",
"keywords": [
"synthetic lethality",
"cloud computing",
"cancer genomics",
"cancer dependency",
"systems biology",
"functional genomics"
],
"content": "Introduction\n\nThe concept of synthetic lethality (SL) refers to interactions between two genes in which loss of function of either gene alone does not impair cell viability, whereas inhibition of both genes is lethal (O’Neil et al., 2017). In the context of anticancer therapy, two genes are synthetic lethal if mutation of either alone is compatible with viability but mutation of both leads to death (Kaelin, 2005). It can be extended to the broader concept that the alteration of one gene is compatible with viability, but the alteration of both leads to death or lower viability. These interactions are attractive for designing cancer therapies, as targeting a gene whose synthetic lethal partner is permanently inactivated in cancer cells but exhibits wild-type expression in healthy cells should selectively kill cancer cells. The synthetic lethal interaction (SLI) between the poly (ADP-ribose) polymerase (PARP) genes and BRCA deficiency (functional loss of either BRCA1 or BRCA2) is the first successful clinical application of the SL concept (Fong et al., 2009; Lord & Ashworth, 2017). Subsequent functional screens have proposed other synthetic lethal pairs, including the SWI/SNF chromatin remodeling complex members SMARCA2-SMARCA4 (Hoffman et al., 2014) and ARID1A-ARID1B (Helming et al., 2014), as well as the Werner syndrome RecQ-like helicase (WRN) gene in MYC overexpressing cancers (Moser et al., 2012) and microsatellite unstable cancers (Chan et al., 2019; Kategaya et al., 2019; Lieb et al., 2019). Although SL-based therapeutics are promising, other drugs for clinical use designed using an SL-based rationale are still under development. There is, therefore, a continued need to discover additional synthetic lethal gene pairs and to develop automated methods that use various data types to predict clinically relevant synthetic lethal pairs that can be nominated for further testing and therapeutic development (Huang et al., 2020).\n\nFunctional screening using siRNA/shRNA technology or, more recently, CRISPR-based targeting libraries, is a leading method of SLI discovery (O’Neil et al., 2017). However, identifying robust synthetic lethal gene pairs is challenging, in part due to biological factors such as genetic and epigenetic heterogeneity and incomplete penetrance, i.e. context-dependent SL (Chan et al., 2010; Henkel et al., 2019; Nijman & Friend, 2013; Ryan et al., 2018). To complement functional screening efforts, multiple computational prediction strategies have been pursued (reviewed by (O’Neil et al., 2017)). Early approaches inferred SLIs in humans via ortholog mapping based on genetic interaction networks from experimentally-tractable model organisms such as Saccharomyces cerevisiae (Conde-Pueyo et al., 2009; Kirzinger et al., 2019; Srivas et al., 2016) and Mus musculus (Gurley & Kemp, 2001). Alternative strategies rely on the integrated analysis of multi-omics profiling and functional screening of patient-derived or cancer cell line-based datasets to predict SLIs. These approaches use statistical and/or heuristic methods, such as implicating SL gene pairs via mutually exclusive loss-of-function mutations, shared pathways or protein complex membership (Das et al., 2019; Jerby-Arnon et al., 2014; Lee et al., 2018; Liany et al., 2020; Wappett et al., 2016; Ye et al., 2016). Furthermore, to facilitate interactive exploration of predicted SLIs, several web portals or SLI databases have been published, such as Syn-Lethality (Li et al., 2014), SynLethDB (Guo et al., 2016), the Synthetic Lethality BioDiscovery Portal (SL-BiodP) (Deng et al., 2019), the Cancer Genetic Interaction Database (CGIdb) (Han et al., 2019), and, more recently, SynLeGG (Wappett et al., 2021). These tools present pre-computed synthetic lethal pairs based on the most comprehensive datasets available at the time of publication. This necessarily excludes potential SLIs discoverable by either algorithmic advances or developments in functional screening technologies in terms of scope and throughput. Additionally, there is limited flexibility to explore the existing set of putative SLIs or to change any parameters in the prediction algorithms to better understand how the SL inference was made.\n\nThere is significant complexity in these prediction approaches because of the need to manage the amount of data on which predictions are based, and the need to select the most appropriate datasets and tools without objective criteria to determine how well any given approach performs. Here we provide a cloud-based framework, Synthetic Lethality Cloud (SL-Cloud), that enables the inference of SL gene pairs from multiple prediction approaches simultaneously on the same datasets. As compared to other current computational approaches for SLI prediction, we provide customized scripts and a facile connection to large public data resources, simplifying the use of publicly available data that can be repurposed for SLI prediction. SL-Cloud is a new component of the Institute for Systems Biology Cancer Gateway in the Cloud (ISB-CGC) resource, a data science infrastructure that provides secure access to a large, comprehensive, and expanding collection of cancer research data (Reynolds et al., 2017). The software draws on and adds to the ISB-CGC resources, enabling the identification of potential SL gene pairs for a specific cancer of interest. We present here an overview of our implementation of the SL-Cloud resource, as well as use cases that showcase the utility of the resource for SL research.\n\n\nMethods\n\nSL-Cloud aggregates commonly used public data resources relevant for SL inference and integrates them with analysis workflows to infer SLIs by leveraging the cloud-based resources stored on the ISB-CGC platform (Figure 1). These three components (summarized below) represent an ecosystem that integrates software and data to enable the large-scale prediction of SLIs from existing cloud-hosted datasets.\n\nSchematic overview of the computational workflows and data resources aggregated in this framework to facilitate investigation of synthetic lethal interactions. Users with specific research questions (top-left) reuse inference workflows from the provided scientific computing notebooks to query public cancer genomics datasets from the vast resources provided by the ISB-CGC and additional datasets pre-processed in SL-Cloud. Three candidate synthetic lethal pair inference workflows were implemented, including the DAta-minIng SYnthetic lethality identification (DAISY), mutation-dependent synthetic lethality prediction (MDSLP), and conserved genetic interaction (CGI) workflows. Different data resources and data types were used for different inference workflows. For example, the DAISY and MDSLP workflows rely on statistical testing over the multiple omics data and functional screening data such as CRISPR and shRNA datasets for human cancers, whereas the CGI workflow is based on an ortholog mapping of the SL interactions identified in yeast.\n\nISB-CGC, a part of the National Cancer Institute (NCI) Cancer Research Data Commons, hosts derived data in Google Cloud Platform BigQuery tables, providing gene expression, mutation, copy number alteration, methylation, protein levels and other molecular characteristics for a broad range of cancers, such that these datasets can be rapidly analyzed using the power of cloud computing (Bleich, 2022; Reynolds et al., 2017). Google BigQuery is a columnar data warehousing solution that provides fast access through Structured Query Language (SQL)-based queries. The ISB-CGC resource contains over 1000 distinct derived data tables that are findable through both a BigQuery Table Search interface as well as through the native Google BigQuery interfaces. On this platform, users can analyze multiple large-scale datasets solely or in combination with their own datasets while circumventing the need to download and maintain a local copy of these petascale datasets, or to perform extensive data management tasks. We have designed SL-Cloud as a component of ISB-CGC so that we can leverage ISB-CGC’s broad availability, co-location of robust computational resources, and democratized access to raw and derived cancer research data. SL-Cloud allows cancer researchers access to key datasets and workflows to enable the identification of potential synthetic lethal gene pairs for a specific cancer of interest.\n\nA key feature of SL-Cloud is the aggregation of key cancer multi-omics datasets for SL inference in a single framework that facilitates integrative analysis. For SL-Cloud, we created and mined the relevant large-scale, publicly available multi-omics and functional screening datasets on ISB-CGC such as The Cancer Genome Atlas (TCGA) (Hutter & Zenklusen, 2018) patient-level data on somatic mutations, gene expression, and copy number alterations across 33 cancer types, (Figure 1) (for details see Table 1, which includes the URLs for specific resources). We identified additional public data resources that were pertinent for SL inference but previously unavailable on ISB-CGC. These datasets include a genetic interaction dataset TheCellMap derived from model organism interaction screens (Costanzo et al., 2016), and human pan-cancer cell line molecular characterization and functional screening datasets, primarily from the Cancer Cell Line Encyclopedia (CCLE) and the Cancer Dependency Map (DepMap) initiative (Dempster et al., 2019; Ghandi et al., 2019; McFarland et al., 2018; Meyers et al., 2017). Building on the infrastructure of the ISB-CGC, we established a new SL-focused cloud resource within ISB-CGC that incorporates the most relevant datasets for SLI prediction (Table 1).\n\nWe implemented SLI inference workflows and distributed them as a set of Jupyter notebooks that use functions from Python scripts provided with this resource. The notebooks offer code optimization and integration with the ISB-CGC through the BigQuery interface to access the relevant pre-processed large-scale cancer genomics datasets described above through embedded SQL queries. Importantly, the workflows can be tailored to individual cancer researcher needs by copying the existing code and optimizing it to their specific use case. We present here a brief overview of three example workflows implemented (for technical details see accompanying documentation on the project GitHub page, the url is available in the Data and software availability section).\n\nThe DAta-minIng SYnthetic lethality identification workflow (DAISY): This previously published workflow is re-implemented using up-to-date, large-scale data resources as described above (Figure 1; Table 1) (Jerby-Arnon et al., 2014). DAISY applies multiple inference procedures that include:\n\ngenomic survival of the fittest (SoF): the detection of infrequently co-inactivated gene pairs by using somatic mutation, copy number alteration and gene expression data\n\nfunctional examination (FunEx): the identification of gene pairs in which inactivation or over-activation of one gene induces essentiality of a partner gene - using functional screening data\n\npairwise gene co-expression: the detection of significantly positively correlated gene pairs - thereby implicating genes in related biological functions\n\nInference of synthetic dosage lethality (SDL), whereby overactivation of one gene causes its interaction partner to become essential for cell viability, is also implemented in DAISY. DAISY SL predictions are gene pairs that are found by all three inference modules. Each individual module can also provide evidence of SL potential independently. The workflow, as we have implemented it, enables users to list predicted synthetic lethal pairs from each workflow, for each dataset, and aggregate them or use them independently. The DAISY workflow also enables users to perform pan-cancer or tissue type-specific analyses by tuning to the specific biological question being examined.\n\nMutation-dependent synthetic lethality prediction (MDSLP): This workflow combines mutation and functional screening data to infer SL pairs from cancer cell line data. The MDSLP workflow is based on the rationale that, for tumors with mutations that have an impact on protein expression or structure (functional mutation), the knockout effects or inhibition of a partner target gene show conditional dependence for the mutated molecular entities (Figure 1). Leveraging the public cancer cell line datasets including gene mutation data from CCLE, and functional screening data generated by either shRNA or CRISPR technology from DepMap (Dempster et al., 2019; Ghandi et al., 2019; McFarland et al., 2018; Meyers et al., 2017), we integrated these data modalities to evaluate mutation-based conditional dependence. This workflow enables users to statistically test whether the knockout or knockdown effects for one gene will be altered if another gene is mutated in specific contexts, such as in pan-cancer, or tumor type-specific cell lines. The increase in gene knockout or knockdown sensitivity provides evidence to support potential SLIs.\n\nConserved genetic interaction (CGI) workflow: We implemented this workflow based on published methods described in (Srivas et al., 2016). The CGI workflow leverages cross-species conservation to infer experimentally derived SLIs in yeast to predict relevant synthetic lethal pairs in humans. For SL-Cloud, we downloaded and preprocessed TheCellMap dataset, the most comprehensive S. cerevisiae genetic interaction network inferred from synthetic genetic array (SGA) screens from (Costanzo et al., 2016) (see details in Table 1). Genetic interactions are inferred if the combined effect of a double mutant on cell viability differs from that of the combination of single mutant effects. SLIs are defined in this context as negative genetic interactions in which the cell viability of a double-mutant yeast colony is lower than that of the respective single-mutant colonies. We provide the inferred SLIs in humans by yeast-to-human ortholog mapping.\n\nSL-Cloud workflows are implemented in a set of Python notebooks that can be edited and run via a Jupyter notebook interface. All the requirements are specified in the respective Jupyter notebooks. Users can also access and run the SL-Cloud workflows in mybinder.org, a platform that allows users to run the implementations without installing any libraries or downloading code from a GitHub repository (see setup and operation instructions here).\n\n\nUse cases\n\nSL-Cloud facilitates custom analyses demonstrated in workflows for particular research questions or disease contexts. Additionally, this framework provides extensibility by virtue of the modular design of the base framework shown in Figure 1. End-users can combine high-quality public data with their own laboratory-generated data to extend integrated analyses more easily without the need to download and pre-process large-scale public cancer genomics data. In the following sections, we describe specific use cases.\n\nSynthetic lethality between BRCA1/2 and PARP1/2 is well documented and is the rationale behind the design of PARP inhibitors such as olaparib, rucaparib, and niraparib (Ashworth & Lord, 2018). These agents are approved for treating BRCA-mutated ovarian cancer and advanced breast cancer. To perform an in silico validation analysis of this well-established SLI, we applied MDSLP to gene mutation and functional screening data from pan-cancer cell lines (Dempster et al., 2019; Ghandi et al., 2019; McFarland et al., 2018; Meyers et al., 2017). As shown by the MDSLP-shRNA workflow and consistent with our expectations, functional mutations of BRCA2 showed significant sensitivity to PARP1 knockdown (two-sided t-test, P < 0.01, FDR < 0.1). We applied the same workflow to gene essentiality data derived from CRISPR screens, but did not find this expected interaction. The MDSLP workflow using CRISPR-derived datasets revealed that the functional mutation of BRCA2 shows a synthetic lethal partnership with PARP2 (P < 0.01, FDR < 0.05). shRNA-derived and CRISPR-derived BRCA2 synthetic lethal pairs showed limited overlap (Figure 2A). Only 6.6% (48 out of 729) of the BRCA2-related synthetic lethal pairs nominated from shRNA-derived inference with a threshold of FDR < 0.1 were also predicted using CRISPR essentiality screens. Of the 1433 potential partner genes predicted by any of the resources, only 48 partner genes were predicted by two resources (Figure 2B). Of these, WRN, TSC2, RPL22L1 showed the most significance with both CRISPR-derived and shRNA-derived inference (FDR < 0.01 for both inference procedures).\n\nA. Network-based representation of predictions generated by the mutation-dependent synthetic lethality prediction (MDSLP) using either shRNA (upper panel, orange) or CRISPR (lower panel, blue) functional screening datasets. B. Potential synthetic lethal partners of BRCA2 as predicted by both the CRISPR and shRNA functional screening data. Each node represents a gene, and edges represent potential synthetic lethal interactions. The node color encodes overlap between the gene-dependency assay type, with yellow representing synthetic lethal pairs predicted in both the CRISPR data and the shRNA functional screening data. The node size and font size indicate the strength of the statistical relationship, with high-confidence pairs having larger node sizes or font sizes. FDR levels: < 0.01 (largest), < 0.05 (median), and <0.1 (smallest).\n\nInterestingly, we did not predict any BRCA2-related synthetic lethal pairs from the other two workflows implemented in SL-Cloud. BRCA2 has no yeast homolog and, therefore, conserved interactions could not be inferred by the CGI workflow. DAISY nominated no synthetic lethal partners for BRCA2 with its default settings but predicted potential BRCA1-PARP1/2 SLIs across all three of its component inference modules with non-default parameter settings. Both gene pairs, BRCA1-PARP1 and BRCA1-PARP2, showed statistically significant co-expression, with their correlation coefficients ranging from 0.26 to 0.59 across patient-derived and cancer cell line datasets [Figure 3A(i,ii) and 3B(i,ii)] (P < 0.01). In addition, we found statistical support for a BRCA1 and PARP1/2 SLIs by the SoF inference procedure [Figures 3A(iii) and 3B(iii,iv)] (P < 0.05), whereas the FunEx module found statistical support for a BRCA1-PARP1 SLI [Figure 3A(iv)] (P < 0.01), but not for a BRCA1-PARP2 SLI, based on the cancer cell line gene-dependency CRISPR or shRNA datasets. In summary, the BRCA1-PARP1 interaction was supported by all three DAISY inference modules, whereas only two modules supported the BRCA1-PARP2 interaction.\n\nA. Evidence for a BRCA1-PARP1 synthetic lethal relationship by pairwise co-expression in i) Pan-Cancer Atlas and ii) CCLE datasets respectively; iii) by survival of the fittest in in CCLE data; and iv) by functional examination in DepMap CRISPR. B. Evidence for a BRCA1-PARP2 SL relationship by pairwise co-expression in i) Pan-Cancer Atlas and ii) CCLE datasets and by survival of the fittest in iii) Pan-Cancer Atlas and iv) CCLE datasets. R, Spearman correlation coefficient; p, P value by the one-sided Wilcoxon rank-sum test; * P < 0.05; ** P <0.01; **** P < 0.0001.\n\nThis example demonstrates how SL-Cloud facilitates the exploration of the SLIs for a particular gene by using orthogonal SLI prediction workflows and multiple datasets to assess the stability and reproducibility of particular SLIs of interest. For the established SLI between BRCA deficiency and PARP1/2 enzymes, we saw variation in the output of multiple prediction approaches and datasets in confirming this bona fide SLI. These analyses highlight some of the challenges related to SL prediction, including unaccounted for variation resulting from differences in the technology and size of the datasets used to make the SL prediction, and the implicit or explicit assumptions made by the underlying analytical approaches. This example shows how SL-Cloud can be applied to enable researchers to explore a particular SLI of interest in different datasets or using different prediction approaches.\n\nSLI partners tend to form functional interaction networks (Costanzo et al., 2016; Jerby-Arnon et al., 2014). For example, Ku et al. reported that synthetic lethal screen hits are more robust at the pathway rather than at the gene level (Ku et al., 2020). To demonstrate pathway-based SLI discovery, we analyzed SLI-related genes in the DNA damage and repair (DDR) pathway. DDR deficiency due to loss-of-function alterations by mutation, deletion, or epigenetic silencing is prevalent across lineages affecting approximately 33% of all cancers in TCGA (Knijnenburg et al., 2018). Impaired DDR leads to genomic instability, and tumors exhibiting DDR loss are prone to DNA-damaging agents and, therefore, potentially vulnerable to inhibitors that target compensatory DDR pathways via a synthetic lethal mechanism (Lord & Ashworth, 2012). Using a well-curated set of 276 genes annotated for involvement in DNA damage repair from (Knijnenburg et al., 2018) we predicted synthetic lethal partners from the three workflows described above (Figure 1).\n\nConsistent with our expectations, different SL prediction approaches led to a diverse set of predicted SLIs. Each workflow identified more than 1,000 synthetic lethal/synthetic dosage lethal partner genes, except for CGI, which identified only 67 synthetic lethal partner genes. Predicted SLI gene sets largely did not overlap; however, functional enrichment analysis showed shared pathway involvement in the interactions identified (Figure 4A). In particular, we found significant KEGG pathway enrichment in synthetic lethal partner genes for the cell cycle, RNA metabolism, splicing machinery, chromatin organization, and transcriptional regulatory pathways (FDR < 0.05). Interestingly, several of these results are broadly related to genomic stability maintenance, and as such, confirm previously published reports from Ku et al., 2020 and others that genes involved in SLIs tend to belong to related pathways. Gene ontology biological processes (GOBPs) enriched by synthetic lethal partner genes vary, but a clustering analysis based on hierarchical structure and semantic similarity of GOBPs showed the synthetic lethal partner genes identified via the different approaches were associated with similar biological processes (Figure 4B). A clustering analysis summarized 350 GOBPs that were initially identified via four approaches into 25 representative GOBP groups. The 25 GOBP groups are associated with synthetic lethal partner genes identified by at least two workflows, and their biological functionality is mirrored by the pathway enrichment results presented above, with enrichment in genes involved in the cell cycle, transcriptional regulation, chromatin organization, and the DNA damage response. In summary, we have demonstrated that pathway-based SL prediction is easily implemented in this framework and can quickly generate useful insights beyond the single-gene level.\n\nHeatmaps depicting A. the KEGG or REACTOME pathway and B. Gene Ontology Biological Process (GOBP) enrichment for predictions made using four different approaches (columns). Increasing color intensity represents increasing statistical significance (P < 0.05, calculated by a hypergeometric test) for enrichment. Pathways or GOBPs were labeled if they were enriched by synthetic lethal partner genes identified by at least two prediction approaches. The redundant GOBPs were further reduced by REVIGO. The 398 GOBPs enriched by at least one approach were reduced to 172 GOBPs based on their semantic similarities, and then summarized into 27 representative groups whose enrichment significance is represented in the heatmap. Clustering analysis was performed for GOBPs inside and outside of the 27 representative groups, separately. The red line down the left side of panel B indicates the separation between the clustering analyses. DAISY, data mining synthetic lethality identification workflow; MDSLP-CRISPR, mutation-dependent synthetic lethality prediction workflow with CRISPR; MDSLP-shRNA, mutation-dependent synthetic lethality prediction workflow with shRNA; CGI, conserved synthetic lethal interactions from yeast screens.\n\nAn overlooked factor that can affect reproducibility of SLIs is context dependence. Genetic background, epigenetic cell state, and tissue type can influence synthetic lethal genetic interactions (Nijman & Friend, 2013; Ryan et al., 2018). We show that the MDSLP workflow and our re-implementation of the DAISY algorithm can be applied to restricted subsets of the underlying data that represent samples or cell lines arising from the same cancer type. The rationale behind this type of analysis is that samples from the same cancer type could represent similar cellular origins, having a characteristic genetic interaction network that is tissue-type specific.\n\nTo illustrate this principle, we investigated the previously reported SLI between ARID1A and ARID1B. Functional loss of ARID1B is a specific vulnerability in ARID1A-mutated cancers, as it affects the composition of the SWI/SNF complex (Helming et al., 2014). We applied MDLSP and DAISY to predict synthetic lethal partners for ARID1A. Via the MDLSP workflow, we found statistical evidence of differential dependency for ARID1B between ARID1A-mutated and wild-type cell lines in various cancer types, suggesting the potential for a SLI between the two genes across tissue types (Figure 5A). Similar to our findings with the BRCA-related synthetic lethal partners, we also saw differences in the strength of the relationship based on whether ARID1B was knocked down via shRNA or knocked out using the CRISPR-Cas9 system. As there is strong and compelling evidence for this SLI, we find support for the interaction occurring across multiple cancer types, even if the evidence comes from shRNA-derived or CRISPR-derived dependency datasets alone.\n\nA. Evidence for synthetic lethality generated from the mutation-dependent synthetic lethality prediction (MDSLP) workflow as applied to cancer cell line dependency datasets when comparing the gene dependency scores (effects) for the shRNA and CRISPR datasets between the ARID1A-mutated group and wild-type group for different cancer types and across all cell lines (pan-cancer analysis). The threshold for statistical significance is FDR < 0.05. B. Statistical evidence for a synthetic lethal relationship between ARID1A and ARID1B from the DAta mIning SYnthetic lethality identification workflow (DAISY), with the results for each inference module being represented in the columns. Column heatmaps summarize the datasets used for each procedure. An asterisk (*) indicates that a test passed the FDR threshold (0.05); gray shading represents an invalid test or a lack of data availability. Gray shading represents an invalid test. C. Heatmap visualization depicting the Spearman correlation between ARID1A and ARID1B for the annotated cancer type for a patient derived sample (Pan-Cancer Atlas) or cancer cell line (CCLE) (rows) across different cancer types (columns).\n\nDAISY does not predict an SLI between ARID1A and ARID1B when applied strictly, that is, when the requirement is set for statistically significant evidence across all three DAISY inference modules (Figure 5B). However, when considering each module individually, we see strong support for the interaction, with strong positive correlation (Spearman ρ in the range [0.3 to 0.77]) between these two genes across almost all cancer types considered (Figure 5B, C). Similar to the findings with MDSLP, we found evidence for the interaction between these two genes in ovarian cancer by using the functional examination module applied on shRNA-derived dependency dataset. This is unsurprising, as the underlying rationale for the DAISY functional examination inference procedure and MDSLP inference strategy are quite similar, and both approaches are applied to the same dataset. We found no statistical support for the interaction via genomic SoF inference, which may be explained by the fact that neither of those genes is inactivated by recurrent focal deletions that underpin that inference module.\n\nThis illustrative example showcases the flexibility of the SL-Cloud framework in that it is relatively easy to compare the results of different SL prediction approaches, while varying algorithmic parameters, using the same or different data types, or to restrict analysis to a given tumor type for further elucidation of context specificities in SL.\n\n\nDiscussion\n\nThe synthetic lethality concept presents a systematic framework with which to identify and nominate potential targets for cancer treatments (Hartwell et al., 1997) Although the SL concept offers a compelling rationale to inform drug target identification, systematically testing all potential SLIs in a given tissue or disease context is experimentally intractable. Therefore, there is a continued need to develop reproducible computational inference and prioritization frameworks that make it easier to nominate the most likely SLIs for experimental follow-up or to aid in functional screen design.\n\nHere we presented a new component of ISB-CGC, SL-Cloud, that brings together computational workflows alongside large-scale datasets via cloud infrastructure to facilitate highly scalable and customizable SL analyses demonstrated through these workflows. The current implementation focuses on axes such as prevalence of genomic alterations in human samples or interactions limited to specific pathways. However, the conceptual design of the framework allows for continued modular development and extensibility. Overall, SL-Cloud offers an ensemble of methods and datasets that enables users to collate evidence for SLIs more easily, leveraging both the richness of existing publicly available datasets and facilitating the integration of smaller user-generated custom or private datasets into the same analysis framework. We anticipate that this resource will enable investigators to look for corroborating evidence for synthetic lethal genetic interactions with therapeutic potential and to explore such interactions in specific biological contexts.\n\n\nData availability\n\nAll datasets supporting the current study and relevant to SL inference are hosted on the ISB-CGC (Reynolds et al., 2017) in existing Google BigQuery tables (Table 1).\n\nProject documentation describing how to access and use this resource are available on the project GitHub page: https://zenodo.org/badge/latestdoi/476040191.\n\n\nSoftware availability\n\nSoftware available from: https://github.com/isb-cgc/SL-Cloud-F1000/releases/tag/F1000\n\nArchived software as at time of publication: https://doi.org/10.5281/zenodo.6400076\n\nLicense: Apache License 2.0.",
"appendix": "Acknowledgments\n\nThe results published here are fully or partially based upon data generated by the TCGA Research Network and the Cancer Target Discovery and Development (CTD2) Network established by the National Cancer Institute’s Office of Cancer Genomics. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. The authors would like to thank Dr. Fabian Seidl, Lauren Hagen, Deena Bleich, and Dr. David Gibbs of the ISB-CGC team for their support, and Dr. William C. Hahn for facilitating access to and enabling the cloud-based redistribution of the Cancer Dependency Map data. We thank Dr. Arko Dasgupta and Russel Mosser from Fred Hutchinson Cancer Research Center for their constructive feedback and Andrew Baumgartner from the Institute for Systems Biology for his suggestions on the project GitHub page, as well as many of our other colleagues and CTD2 collaborators for helpful discussions and critical manuscript feedback. The authors thank Keith A. Laycock, PhD, ELS, for scientific editing of the original version of the manuscript. We dedicate this paper to the memory of Dr. Daniela S. Gerhard. We are grateful for her constant encouragement, scientific suggestions and support for this project.\n\n\nReferences\n\nAshworth A, Lord CJ: Synthetic lethal therapies for cancer: what’s next after PARP inhibitors? Nat Rev Clin Oncol. 2018; 15(9): 564–576. PubMed Abstract | Publisher Full Text\n\nBleich D: ISB-CGC Cloud Resource: Providing Researchers with Shortcuts to Data Analysis. 2022. 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PubMed Abstract | Publisher Full Text | Free Full Text\n\nKirzinger MWB, Vizeacoumar FS, Haave B, et al.: Humanized yeast genetic interaction mapping predicts synthetic lethal interactions of FBXW7 in breast cancer. BMC Med Genomics. 2019; 12(1): 112. PubMed Abstract | Publisher Full Text | Free Full Text\n\nKnijnenburg TA, Wang L, Zimmermann MT, et al.: Genomic and molecular landscape of DNA damage repair deficiency across The Cancer Genome Atlas. Cell Rep. 2018; 23(1): 239–254.e6. PubMed Abstract | Publisher Full Text | Free Full Text\n\nKu AA, Hu HM, Zhao X, et al.: Integration of multiple biological contexts reveals principles of synthetic lethality that affect reproducibility. Nat Commun. 2020; 11(1): 2375. PubMed Abstract | Publisher Full Text | Free Full Text\n\nLee JS, Das A, Jerby-Arnon L, et al.: Harnessing synthetic lethality to predict the response to cancer treatment. Nat Commun. 2018; 9(1): 2546. PubMed Abstract | Publisher Full Text | Free Full Text\n\nLi XJ, Mishra SK, Wu M, et al.: Syn-lethality: an integrative knowledge base of synthetic lethality towards discovery of selective anticancer therapies. Biomed Res Int. 2014; 2014: 196034. PubMed Abstract | Publisher Full Text | Free Full Text\n\nLiany H, Jeyasekharan A, Rajan V: Predicting synthetic lethal interactions using heterogeneous data sources. Bioinformatics. 2020; 36(7): 2209–2216. PubMed Abstract | Publisher Full Text\n\nLieb S, Blaha-Ostermann S, Kamper E, et al.: Werner syndrome helicase is a selective vulnerability of microsatellite instability-high tumor cells. Elife. 2019; 8: e43333. PubMed Abstract | Publisher Full Text | Free Full Text\n\nLord CJ, Ashworth A: The DNA damage response and cancer therapy. Nature. 2012; 481(7381): 287–294. PubMed Abstract | Publisher Full Text\n\nLord CJ, Ashworth A: PARP inhibitors: synthetic lethality in the clinic. Science. 2017; 355(6330): 1152–1158. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMcFarland JM, Ho ZV, Kugener G, et al.: Improved estimation of cancer dependencies from large-scale RNAi screens using model-based normalization and data integration. Nat Commun. 2018; 9(1): 4610. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMeyers RM, Bryan JG, McFarland JM, et al.: Computational correction of copy number effect improves specificity of CRISPR-Cas9 essentiality screens in cancer cells. Nat Genet. 2017; 49(12): 1779–1784. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMoser R, Toyoshima M, Robinson K, et al.: MYC-driven tumorigenesis is inhibited by WRN syndrome gene deficiency. Mol Cancer Res. 2012; 10(4): 535–545. PubMed Abstract | Publisher Full Text | Free Full Text\n\nNijman SMB, Friend SH: Cancer. Potential of the synthetic lethality principle. Science. 2013; 342(6160): 809–811. PubMed Abstract | Publisher Full Text\n\nO’Neil NJ, Bailey ML, Hieter P: Synthetic lethality and cancer. Nat Rev Genet. 2017; 18(10): 613–623. PubMed Abstract | Publisher Full Text\n\nReynolds SM, Miller M, Lee P, et al.: The ISB Cancer Genomics Cloud: a flexible cloud-based platform for cancer genomics research. Cancer Res. 2017; 77(21): e7–e10. PubMed Abstract | Publisher Full Text | Free Full Text\n\nRyan CJ, Bajrami I, Lord CJ: Synthetic lethality and cancer - penetrance as the major barrier. Trends Cancer Res. 2018; 4(10): 671–683. PubMed Abstract | Publisher Full Text\n\nSrivas R, Shen JP, Yang CC, et al.: A network of conserved synthetic lethal interactions for exploration of precision cancer therapy. Mol Cell. 2016; 63(3): 514–525. PubMed Abstract | Publisher Full Text | Free Full Text\n\nWappett M, Dulak A, Yang ZR, et al.: Multi-omic measurement of mutually exclusive loss-of-function enriches for candidate synthetic lethal gene pairs. BMC Genomics. 2016; 17: 65. PubMed Abstract | Publisher Full Text | Free Full Text\n\nWappett M, Harris A, Lubbock ALR, et al.: SynLeGG: analysis and visualization of multiomics data for discovery of cancer 'Achilles Heels' and gene function relationships. Nucleic Acids Res. 2021; 49(W1): W613–W618. PubMed Abstract | Publisher Full Text | Free Full Text\n\nYe H, Zhang X, Chen Y, et al.: Ranking novel cancer driving synthetic lethal gene pairs using TCGA data. Oncotarget. 2016; 7(34): 55352–55367. PubMed Abstract | Publisher Full Text | Free Full Text"
}
|
[
{
"id": "144718",
"date": "18 Aug 2022",
"name": "Jane Usher",
"expertise": [
"Reviewer Expertise Fungal biology",
"molecular biology",
"microbiology",
"chemogenomic and gentic interaction screens."
],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nIn this paper by Tercan et al., the authors present a synthetic-lethal cloud-based component of the ISB-CGC that can provide an upgraded and integrated framework of cloud-based data resources and workflows to enable and enhance the prediction of synthetic lethal interactions in cancer tissue networks. This tool is devised in order to address the challenge of pre-processing large datasets from multiple sources and the availability to perform multiple prediction comparisons. The SL-cloud allows users to customise inputs and then test predictions of SLIs approaches across multiple datasets and add in new studies. This tool currently is focused on use by cancer biology researchers in support of the development of potential drug targets, such as the case for BRAC1/2 and PARP1.\nIt would interesting if the authors could expand more in the discussion or provide an example that is not already shown in previous publications of how this tool may be modified to other organisms, such as fungal pathogens where a slue of SLI studies are undertaken with many times laborious workflows.\nOverall, this was an exciting paper to read and very written with a timely concept.\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": "9125",
"date": "22 Dec 2022",
"name": "Nyasha Chambwe",
"role": "Author Response",
"response": "We added the following text to the Discussion section: “From this perspective, SL-Cloud can be used for predicting SLIs for diseases other than cancer, such as viral and fungal infectious diseases. The conserved genetic interaction (CGI) workflow could be used as is. The computational approaches we proposed can be used with omics and CRISPR datasets that are from fungi/virus infected samples and replacing inactive/mutant genes with genes whose activity or expression is affected by the infection.”"
}
]
},
{
"id": "151441",
"date": "25 Oct 2022",
"name": "Nishanth Ulhas Nair",
"expertise": [
"Reviewer Expertise Computational biology",
"cancer",
"genetic interactions"
],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nIn this project, the authors present SL-Cloud which provides an integrated framework of cloud-hosted data resources and curated workflows to enable facile prediction of synthetic lethal interactions. This tool may be very useful.\nI have a few minor comments for the authors to consider.\n\nIt is not clear why DAISY and MDSLP were chosen for implementation when many other prediction pipelines exist. Perhaps the authors can clarify.\n\nMany of the computational methods to identify synthetic lethal (SL) interactions may have false positives or false negatives. This needs to be mentioned as a limitation in the paper, so that the readers are aware of this.\n\nThere are some experimental double gene knockout datasets in human cancer cell lines that could be used to detect experimental SL interactions. For example: Horlbeck M, Xu A, Wang M, Bennett N, et al. (2018)1\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": "9126",
"date": "11 Jan 2023",
"name": "Nyasha Chambwe",
"role": "Author Response",
"response": "Comment: It is not clear why DAISY and MDSLP were chosen for implementation when many other prediction pipelines exist. Perhaps the authors can clarify The following paragraph has been added to the Discussion section: “In this resource we have implemented the workflows that are widely used and are the basis for other algorithms. We have reimplemented the CGI workflow because most of the computational approaches start with identifying SLIs in yeast and mapping these to their human orthologs (Thompson et al., 2015). The DAISY workflow is comprehensive, data-driven and performs SLI search from several different aspects using multiomics and screening data. It comprises individual inference modules and is extensible to handle other evidence, such as co-pathway membership, giving researchers an example of how to analyze and integrate different datasets. DAISY has been cited extensively and similar assumptions to those in DAISY have been implemented by other authors (Das et al., 2019; Liu et al., 2022; Sinha et al., 2017). Sinha et al., 2017 claim that the DAISY algorithm is restrictive in the sense that DAISY uses a limited variety of inactivating mutations. Liu et al., 2022 points out that DAISY results are non-specific pan-cancer inferences and that it doesn’t provide tissue specific results (this is an example of context sensitivity). Our resource provides a good starting point and ready to use implementations for similar research. The MDSLP workflow reflects one of the most important applications for SL discovery in cancer. Many driver mutations for different cancer types have been characterized and the MDSLP can be used to identify the SL pairs for cancer driver genes, such as PARP1 - BRCA, as shown in Fig 2. The workflow can provide important clues for novel target prediction.” Comment: Many of the computational methods to identify synthetic lethal (SL) interactions may have false positives or false negatives. This needs to be mentioned as a limitation in the paper, so that the readers are aware of this. The following sentences have been added to the Discussion section: “SLIs are context sensitive and the computational methods all have their own assumptions in search for SL pairs. Depending on the context, sample size, cohort heterogeneity and the assumptions that the computational approach relies on, researchers may get false positive and false negative results. It should be added that the very notion of false positive/negative ipso facto requires the existence of a ‘ground truth’, which is in essence contrary to the idea of context sensitivity. Our resource allows researchers to explore different SLI prioritization approaches using a wide range of multiomics and screening data. Such exploration can produce more reproducible and reliable results along with a better understanding of the predictions from different biological perspectives.” Comment: There are some experimental double gene knockout datasets in human cancer cell lines that could be used to detect experimental SL interactions. For example: Horlbeck M, Xu A, Wang M, Bennett N, et al. (2018) We have cited the paper in the proper context, where we mention CRISPR perturbations for SLI identification: “A recent study detects human SLIs based on systematic CRISPRi perturbation of over two thousand gene pairs” sentence has been added in the second paragraph of the Introduction and Horlbeck M, Xu A, Wang M, Bennett N, et al. (2018)"
}
]
}
] | 1
|
https://f1000research.com/articles/11-493
|
https://f1000research.com/articles/11-1567/v1
|
22 Dec 22
|
{
"type": "Research Article",
"title": "Perception of nurses, medical laboratory scientists, and midwives toward coronavirus vaccination in Khartoum State, 2021—a cross sectional study",
"authors": [
"Abrar Tariq Abdelrahman",
"Daffalla A'lam Elhuda",
"Hala Tariq Abdelnabi",
"Abdelfatah Abdellateef Ahmed",
"Ahmed Bakheet Abd Alla",
"Abrar Tariq Abdelrahman",
"Daffalla A'lam Elhuda",
"Abdelfatah Abdellateef Ahmed",
"Ahmed Bakheet Abd Alla"
],
"abstract": "Background: Nurses, medical laboratory scientists and midwives comprise a large portion of healthcare personnel. Healthcare personnel have an important role in guiding and encouraging patients and communities, and showing role modeling behavior. Objective: This study aimed to evaluate and explore the perception of nurses, medical laboratory scientists, and midwives toward coronavirus vaccination. Methods: A descriptive cross-sectional facility-based study was conducted. Data were collected using an online Google form questionnaire. Demographic variables were analyzed using frequencies and percentages. The association between independent variables and the decision of receiving the COVID-19 vaccine were evaluated by binary logistic regression and Chi-square test. Results: In this study, 375 responses were collected, of which 324 (86.4%) were female. The majority of the participants (73.9%) were aged between 20 and 30 years. There were 160 (42.7%) medical laboratory scientists, 145 (38.7%) nurses, and 70 (18.7%) midwives. More than half of the participants (53.6%) accepted receiving vaccination against COVID-19. Results showed a positive correlation of vaccine acceptance with nurses, medical laboratory scientists, and midwives, suggesting that they are more likely to be vaccinated. Conclusion: There was a good perception towards COVID-19 vaccination, as 53.4% of the participants accepted receiving the COVID-19 vaccine, which is a good rate for acceptance. This finding has a positive impact on the whole vaccination process, as the recommendations of medical laboratory scientists, nurses, and midwives affect the behavior of the general population toward vaccination.",
"keywords": [
"coronavirus. COVID-19",
"vaccination",
"perception",
"nurses",
"midwives",
"laboratory scientists"
],
"content": "Introduction\n\nOn December 31, 2019, an unknown etiology caused pneumonia cases in the Hubei Province of China, Wuhan City, and this was reported to the World Health Organization (WHO) by the China Country Office. A new type of coronavirus (novel coronavirus, nCoV) was identified by the Chinese authorities and was isolated on 7 January 2020 as the causative agent [https://www.who.int/emergencies/disease-outbreak-news/item/2020-DON229], [https://reliefweb.int/report/china/novel-coronavirus-china-disease-outbreak-news-12-january-2020?gclid=CjwKCAiAv9ucBhBXEiwA6N8nYNZaGQSiItvL6okVamGegRAKJA4GZvpjroHts6qKoKfhYlu9FvqGMRoCbRQQAvD_BwE].\n\nCoronaviruses are single-stranded RNA viruses categorized into four groups based on the genetic homology: Alpha, Beta, Gamma, and Delta. The betacoronaviruses include SARS-CoV, MERS-CoV 3, and recently the new coronavirus SARS-CoV-2. The name of the virus means crown or halo and originated from the Latin word corona, referring to the crown-like projections on the surface of the virus.1 The family of coronaviruses include viruses with a wide host range and include many types, causing either a mild chest cold or serious infections such as Middle East respiratory syndrome (MERS) and severe acute respiratory syndrome (SARS) [https://www.cdc.gov/coronavirus/2019-ncov/your-health/about-covid-19/basics-covid-19.html]. The official name for the new disease was declared by the WHO as coronavirus disease 2019, abbreviated to COVID-19 on February 11, 2020 [https://www.cdc.gov/coronavirus/2019-ncov/your-health/about-covid-19/basics-covid-19.html].\n\nThe symptoms of COVID-19 vary from mild to moderate respiratory discomfort that may recover without medical intervention. Underlying medical conditions and advanced age may lead to serious illness [https://www.who.int/emergencies/diseases/novel-coronavirus-2019/question-and-answers-hub/q-a-detail/coronavirus-disease-covid-19]. The spreading of COVID-19 continued even after preventive measures such as social distancing, face masks, quarantine and travel restrictions were applied, resulting in serious consequences to life, the economy and health, with the only hope being the development of a successful vaccine.2 Research centers and pharmaceutical companies began the devolvement of vaccines from the emergence of SARS-CoV-2 and the publication of the first genome.3\n\nEarly in December 2020, the first vaccination program began. There have been no less than 13 diverse vaccines administered, including Pfizer/BioNtech Comirnaty, SII/Covishield, AstraZeneca/AZD1222, Moderna COVID-19, Janssen/Ad26.COV2.S, and Sinopharm COVID-19 [https://www.who.int/emergencies/diseases/novel-coronavirus-2019/question-and-answers-hub/q-a-detail/coronavirus-disease-(covid-19)-vaccines?adgroupsurvey={adgroupsurvey}&gclid=CjwKCAiAv9ucBhBXEiwA6N8nYFcBFmhpuV-5rCPhQ0zJRZIn4aG7o0T9XyDP9ZmMW1mNsfOnqXxwHRoCUggQAvD_BwE]. These vaccines enable our bodies to recognize and protect against the virus that causes COVID-19. Vaccines may work as mRNA vaccines, protein subunit vaccines, or vector vaccines [https://www.cdc.gov/coronavirus/2019-ncov/vaccines/different-vaccines/how-they-work.html].\n\nPatient acceptance of vaccination is influenced by the usage of the vaccination by healthcare workers, along with the reduction of vaccine hesitancy. Vaccinated healthcare workers influence the patient to take the vaccine.4 Additionally, there is a constant correlation between vaccine hesitancy amongst healthcare personnel and that reported in the general population.5 Healthcare workers guide and provide trusted information about the vaccine to the general population, and prevent the spreading of misleading and confusing information.2 The intention of healthcare workers to advocate a vaccine for patients relies on their attitudes and knowledge regarding vaccines.4\n\n\nMethods\n\nThis was a descriptive, analytical cross-sectional facility-based study. The questionnaire was conducted online using a Google form. These forms were sent via social media to obtain the answers from participants.\n\nThe study was carried out in Khartoum State, Sudan.\n\nThe study population consisted of nurses, laboratory scientists, and midwives who worked in Khartoum State.\n\nAny nurse, midwife, or laboratory scientist who worked in Khartoum State was eligible to participate.\n\nOther healthcare workers and those who worked in a different state.\n\nThe sample size was 375, and it was estimated assuming a prevalence of 23.3% of satisfaction regarding the teaching methodology, a 1.96 confidence level, and a sample error of 5%.\n\nA simple random sampling technique was applied for this study. The questionnaire was sent online to collect data from the participants, then the data were collated on an Excel spreadsheet and analyzed using SPSS (RID:SCR_002865).\n\nAn online Google form questionnaire was used to collect the data from the participants.\n\nEthical approval was obtained from the Committee of Medical Laboratory Science, ethical No. (DSR–IEC–04–1–2021). Written and verbal informed consent was taken from participants before starting the study for data collection and publication.\n\nData were extracted into a Microsoft Excel spreadsheet and loaded to SPSS version 22.0 (RID:SCR_002865) for final analysis.\n\n\nResults\n\nIn this study, 375 responses were collected, and, of these, 324 (86.4%) were female and 51 (13.6%) were male. The majority (277, 73.9%) of the participants were aged between 20 and 30 years. There were 160 (42.7%) medical laboratory scientists, 145 (38.7%) nurses, and 70 (18.7%) midwives. More than half of the participants 211 (56.3%) were working in the Department of Microbiology in the Khartoum locality, 247 (65.9%) had reached university level, and 138 (36.8%) of the participants had 5–10 years of work experience.\n\nRegarding work circumstances (Figure 1), the majority of respondents had not worked in an isolation center nor had contact with COVID-19 patients (88% and 76%, respectively). About 40% had already had or had been suspected to have COVID-19. Figure 2 shows the level of knowledge according to participant self-estimation. The highest number of participants obtained their information from social media (Figure 3).\n\nThere was a significant association between the refusal of vaccination and age group, occupation, level of education, and years of experience. Participants who were aged between 20 and 30 years, studied till university, were nurses, and had 1–5 years of experience had the highest rate of refusal. On the other hand, there was no significant relation between refusal of vaccination and sex, place of work, working in isolation centers, engaging with COVID-19 patients, and whether the participant had had a suspected or confirmed case of COVID-19. A total of 51% of participants agreed that manager encouragement and advocating affected the decision of whether to have the vaccine. Binary logistic regression was performed for further analysis, and there was no significant association between the dependent variable (vaccination refusal) and the independent variables, as shown in Table 1. Nurses and participants who were over 40 years old were less likely to refuse the vaccine. In contrast, men were 1.7 times more likely to refuse the vaccine than females (OR 1.7, 95% CI 0.94–3.32) and participants who were aged between 30 and 40 years, had 5–10 years of work experience, and had postgraduate qualifications were more prone to refuse the vaccination (OR 1.3, 95% CI 0.38–4.44; OR 1.5, 95% CI 0.64–3.59; OR 3.0, 95% CI 0.48–19.7, respectively).\n\nTable 2 shows some of the factors that may affect vaccine acceptance and the decision to have the vaccine from the personal perspective of participants; the most agreed factor that may increase acceptance was to increase knowledge about vaccine effectiveness (88%).\n\nThe main cause for refusal was insufficient information about vaccine effectiveness, which was reported by 76 participants (Figure 4).\n\n\nDiscussion\n\nInfectious diseases have been a threat to public health for decades, and the main route to eradicate them is by developing vaccines. COVID-19 has become a global issue that has affected the economy, social life, and many other aspects. Healthcare workers’ thoughts about the COVID-19 vaccine play a crucial role in the acceptance rate in the population because they are regarded as a trustworthy and credited provider of healthcare information to the population.\n\nIn this study, more than half of the participants (53.6%) accepted receiving the COVID-19 vaccine. This result was in line with a study carried out in Saudi Arabia by Barry et al., which evaluated COVID-19 vaccine confidence among healthcare workers and discovered that two-thirds of participants expressed a desire to have a potential COVID-19 vaccine.6 A study in France showed a 77.6% acceptance rate,7 and a study carried out in western India showed that 89.4% of people were prepared to have the COVID-19 vaccine.8 Meanwhile, our findings were inconsistent with the studies carried out in Egypt, with a 21% acceptance rate,2 in Congo with 28%,4 and in the USA with 36%.9 The study's findings showed that there was no significant association between acceptance of the COVID 19 vaccine and sex, but the willingness to receive the vaccine varied.\n\nThese results were similar to a study by Fakonti et al. in Cyprus, which demonstrated that females were more likely than males to accept vaccination.10\n\nRegarding age, older participants (more than 40) were more likely to be vaccinated, consistent with the study by Kumar et al.11 This finding could be interpreted as this age group being more responsive than the other age groups and therefore more likely to accept the vaccine.\n\nFurthermore, participants with 5–10 years’ work experience and with postgraduate qualifications were the least likely to accept vaccination.\n\nIn this study, the results showed a positive correlation of vaccine acceptance with nurses, midwives, and medical laboratory scientists, suggesting that they are more likely to be vaccinated, and their direct contact with COVID patients may lead to higher acceptance of COVID-19 vaccination. In this study, 61% of the participants said they encouraged their family members to be vaccinated, which would increase the acceptance rate. This was in line with a study by Fares et al.,2 and a study by Shekhar et al.,9 whose study revealed that healthcare workers who are vaccinated are more likely to recommend vaccines to family, friends, and their patients.\n\nInsufficient information about vaccine effectiveness was the main reason for vaccine refusal, agreeing with the study carried in western India,8 followed by uncertainty about vaccine effectiveness, and adverse effects and complications after vaccination. This finding could be used to minimize the refusal by providing more accurate and sufficient information and studies about vaccines and their possible adverse effects and complications. The most common information sources used in this study were social media followed by mass media. The refusal of vaccination was significantly associated with knowledge of the COVID-19 vaccine(P value 0.003). Approximately 110 respondents stated that they had great knowledge of COVID-19 vaccinations.\n\nIn terms of the working place, there was no significant association between vaccine refusal and whether participants worked in isolation facilities for COVID-19 or interacted with COVID-19 patients directly.\n\nThere are a number of limitations in this study. There was a sex imbalance, with 324 (86%) participants being female and only 14% being male, which could have had an impact on the outcome. In addition, the study was carried in Khartoum State only so the perception of COVID-19 vaccination may be different in other areas of the country.\n\nFinally, only medical laboratory scientists, nurses, and midwives were included in our sample; therefore, our results cannot be generalized to other healthcare professionals.\n\n\nConclusions\n\nThere was a good perception toward COVID-19 vaccination, as 53.4% of the participants accepted having the COVID-19 vaccine, which is a good rate of acceptance. This finding has a positive impact on the whole vaccination process, as medical laboratory scientists, nurses, and midwives make up a large proportion of the healthcare workers around the country and of the general population, and they have a high impact as their recommendations affect the behavior of the general population toward vaccination. Age, occupation, educational level, and years of experience were significantly associated with vaccination acceptance. Insufficient information about vaccine effectiveness was the main reason for vaccination refusal, which can be corrected by providing more accurate and sufficient information and clinical trials on vaccines to increase the rate of acceptance.\n\nEthical approval was received from the Committee of Medical Laboratory Science, ethical No. (DSR–IEC–04–1–2021).\n\n\nConsent\n\nWritten and verbal consent was obtained from participants before starting the study for data collection and publication.",
"appendix": "Data availability\n\nFigshare: Hala Data dictionary.docx. https://doi.org/10.6084/m9.figshare.21630002.v1. 12\n\nThe project contains the following underlying data:\n\n- Hala Data dictionary.docx (data file headings)\n\n- Hala data.xlsx (raw data)\n\nPerception of nurses, medical laboratory scientist and midwives toward coronavirus vaccination at Khartoum State, 2021. https://docs.google.com/forms/d/e/1FAIpQLSdZDYvP6iDpidFWSurnjfmh73-XBv0waHqnLJ8MXA5l0Lvprg/viewform. 13\n\nThe project contains the following underlying data:\n\n- Original questionnaire\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).\n\n\nReferences\n\nRichman DD, Whitley RJ, Hayden FG: Clinical Virology. 4th ed.Washington:ASM Press;2016.\n\nFares S, Elmnyer MM, Mohamed SS, et al.: COVID-19 vaccination perception and attitude among healthcare workers in Egypt. J. Prim. Care Community Health. 2021; 12: 21501327211013303.\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. Published 2020 Jul 14. PubMed Abstract | Publisher Full Text | Free Full Text\n\nKabamba Nzaji M, Kabamba Ngombe L, Ngoie Mwamba G, et al.: Acceptability of vaccination against COVID-19 among healthcare workers in the Democratic Republic of the Congo. Pragmat. Obs. Res. 2020; 11: 103–109. Published 2020 Oct 29. PubMed Abstract | Publisher Full Text | Free Full Text\n\nNaz H, Cevik F, Aykın N: Influenza vaccination in healthcare workers. Infect. Control Hosp. Epidemiol. 1997; 18(3): 189–194.\n\nBarry M, et al.: Covid-19 vaccine confidence and hesitancy among health care workers: A cross-sectional survey from a MERS-COV experienced nation. PLoS One. 2021; 16(11): e0244415. PubMed Abstract | Publisher Full Text | Free Full Text\n\nDetoc M, et al.: Intention to participate in a COVID-19 vaccine clinical trial and to get vaccinated against COVID-19 in France during the pandemic. Vaccine. 2020; 38(45): 7002–7006. PubMed Abstract | Publisher Full Text | Free Full Text\n\nDara S, Sharma SK, Kumar A, et al.: Awareness, attitude, and acceptability of healthcare workers about COVID-19 vaccination in Western India. Cureus. September 30, 2021; 13(9).\n\nShekhar R, Sheikh AB, Upadhyay S, et al.: COVID-19 Vaccine acceptance among health care workers in the United States. Vaccines. 2021; 9: 119. PubMed Abstract | Publisher Full Text | Free Full Text\n\nFakonti G, Kyprianidou M, Toumbis G, et al.: Attitudes and acceptance of COVID-19 vaccination among nurses and midwives in Cyprus: a cross-sectional survey. Front. Public Health. 2021; 9: 656138. PubMed Abstract | Publisher Full Text | Free Full Text\n\nKumar R, Alabdulla M, Elhassan NM, et al.: Qatar healthcare workers’ COVID-19 vaccine hesitancy and attitudes: a national cross-sectional survey. Front. Public Health. 2021; 9: 727748. PubMed Abstract | Publisher Full Text | Free Full Text\n\nFigshare: Hala Data dictionary.docx. DOI: 10.6084/m9.figshare.21630002.v1\n\nPerception of nurses, medical laboratory scientist and midwives toward coronavirus vaccination at Khartoum State, 2021. http"
}
|
[
{
"id": "190646",
"date": "26 Jul 2023",
"name": "John Unsworth",
"expertise": [
"Reviewer Expertise Vaccination hesitancy and workforce"
],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis is an interesting study cutting across a number of professions. The literature is currently limited and there are a number of systematic reviews and umbrella reviews around vaccination hesitancy amongst health professionals which would be useful to cite.\n\nIn the background, I think you really need to outline why vaccination amongst healthcare workers is important. Vaccination ensures we have sufficient workforce to provide care and it is an important element in terms of preventing infection spread.\n\nIn the methodology section - sample size - this statement appears slightly out of the scope of this work 'regarding the teaching methodology'. This is not a report on a pedagogical intervention so I am unsure why it is included.\n\nI would question your conclusions as 53.4% of the study population accepting vaccination is not good and remains inadequate to prevent large-scale absence, long-term effects from long Covid in the workforce and reducing the spread of infection.\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Partly",
"responses": []
},
{
"id": "190576",
"date": "02 Nov 2023",
"name": "Ewa Kusideł",
"expertise": [
"Reviewer Expertise Statistics"
],
"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'm afraid I have to disagree with nearly all statements which commenting on the essential Table 1 - the only one showing the association between vaccine refusal and selected characteristics.\nI can not agree that „There was a significant association between the refusal of vaccination and age group, occupation, level of education, and years of experience”. For example, in the age 20-30 the proportion of the “refusal group” is 146/277 =0.527=52.7% vs 131/277 =0.473=47.3%. If we compare these groups using the chi-square test or the test of proportion, the p-value is p=0.202, which is not statistically significant.\nMore importantly, the logistic regression results do not confirm any association between the \"refusal of vaccination\" and the rest of the variables. The results of this regression oppose the conclusion that “Participants who were aged between 20 and 30 years, studied till university, were nurses, and had 1–5 years of experience had the highest rate of refusal” .\nThis is not true because 20-30 years group was the reference group (without OR) and simple analysis from the beginning of this description does not confirm that:\nOR for \"1-5 years of experience\" is positive but not significant (p-value 0.562); OR for the university level of education is positive but very small and insignificant (p-value=0.818); OR for \"nurses\" is negative (sic!) and statistically insignificant (p=0.319).\nAdditionally, there are errors in the OR value in Table 1, at least in the case of the “<1 year” and “postgraduate” variables: (1) for the “<1 year” variable, the B coefficient is 0.818 then OR should be 2.266 not 0.914, (2) for the “postgraduate” variable B coefficient is 0.129, so it should give OR value equal to 1.138 not 3.092 as authors show in Table 1.\nFinally, the authors do not realize that when commenting on Table 1: “Nurses (…) were less likely to refuse the vaccine” they contradict themselves by writing at the beginning of the same paragraph: “Participants who were (…) nurses, (…) had the highest rate of refusal”.\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? Yes\n\nAre the conclusions drawn adequately supported by the results? No",
"responses": []
}
] | 1
|
https://f1000research.com/articles/11-1567
|
https://f1000research.com/articles/11-1564/v1
|
22 Dec 22
|
{
"type": "Research Article",
"title": "Predictors of unwillingness or inaccessibility to receive the COVID-19 vaccination among persons with disabilities in Bangladesh",
"authors": [
"Md Zahid Hossain",
"Md Akter Hossain",
"Mohammad Yaqub Al Ansary",
"Veena Raigangar",
"Md Habibur Rahman",
"Ruksana Akter",
"Shameem Ahmed",
"Sharmila Jahan",
"Iqbal Kabir Jahid",
"K. M. Amran Hossain",
"Md Zahid Hossain",
"Md Akter Hossain",
"Mohammad Yaqub Al Ansary",
"Veena Raigangar",
"Md Habibur Rahman",
"Ruksana Akter",
"Shameem Ahmed",
"Sharmila Jahan",
"Iqbal Kabir Jahid"
],
"abstract": "Introduction\nPersons with disabilities (PWDs) are among the most vulnerable communities to suffer the serious consequences of COVID-19, and accepting COVID-19 vaccination is one of the recommended health advisories for them. Unwillingness to receive vaccines is a concerning issue, especially in the countries of Southeast Asia. The study aims to find out the COVID-19 vaccination rate of persons with disabilities (PWDs) in Bangladesh, the rate of unwillingness or inaccessibility of vaccination for PWDs, and predict the possible reasons for unwillingness. Methods\nA descriptive cross-sectional survey of PWDs aged 12 years or more was conducted in 12 rehabilitation centers in Bangladesh between February 2022 and May 2022. A self-developed structured questionnaire on socio-demographic, disability, and health indicators, acceptance and accessibility of COVID-19 vaccines, and knowledge and attitude towards vaccination was used for the survey. The study has been conducted according to the Strengthening the Reporting of Observational Studies in Epidemiology (STROBE) guidelines for a cross-sectional survey. Results\nWith a 69% response rate, 241 PWDs participated in the study. PWDs with a spinal cord injury were the major respondents (62.7%). A total of 186 (77.2%) PWDs had taken one or more doses of COVID-19 vaccines of three doses supplied, and 55 (22.8%) were unwilling to have a vaccination. All vaccine recipients reported good accessibility to vaccination booths. The predictive factors of unwillingness were spinal cord injury type of disability (OR .36, P<.01), people coming to the rehabilitation center from rural areas (OR .44, P<.01), poor knowledge of COVID-19 vaccination (OR .78, P<.01), and dependency on mobility (OR.24, P<.001). Conclusion\nMore than one-fifth of the persons with disabilities aged between 12 and 80 years were unwilling to receive the COVID-19 vaccination despite the accessibility of information and availability of the COVID-19 vaccine, mainly due to poor knowledge and mobility issues.",
"keywords": [
"persons with disabilities",
"Bangladesh",
"COVID-19",
"vaccination"
],
"content": "Introduction\n\nBangladesh was targeted to cover 119,221,953 vaccine administrations for COVID-19, and the Directorate General of Health Services (DGHS) reports the achievement of 82.95 % at the first dose, and 72.94 % at the second dose. Nearly 36% of the population received the third dose of the COVID-19 vaccine.1 Since the global vaccine campaign, the World Health Organization has urged special consideration for persons with disabilities (PWDs) as a priority. According to the Bangladesh Bureau of Statistics (BBS), 7% of the population in Bangladesh is living with some form of disability. There are a scarcity of data about the rate of vaccine administration to PWDs in Bangladesh. Though the percentage of registered individuals for COVID-19 vaccination is approximately 94%, a population-based study reported that one-third of Bangladeshi adults had vaccine hesitancy2; these were mostly men from low-income families who were unsure regarding the effects of the COVID-19 vaccine. The unwillingness to receiving vaccines was 27.4%,3 near the hesitancy margin of 33%.2 This unwillingness was found to be associated with a semi-urban dwelling, low income, and a lower educated population. No data related to the unwillingness towards COVID-19 vaccine was found in PWDs in Bangladesh; however, one study reported that 42.7% of parents of children with a neuro-developmental disability express hesitancy towards receiving the vaccine for their children.4\n\nUnwillingness can be defined as not receiving any doses of vaccine despite there being ability to access and scope for vaccination, or not responding to the call for vaccination5, while hesitancy is doubtfulness of receiving vaccine.2 In the UK, 4% of the population was hesitant or uncertain, and another 4% were unwilling to receive the COVID-19 booster dose.5 The former study4 states that people might have poor compliance with guidelines to vaccination; this was particularly seen in vulnerable communities who were unwilling to receive the COVID-19 vaccination. The WHO and UNICEF policy brief states that PWDs are a part of the vulnerable community that needs to be protected through effective public vaccine campaigns. A study in the United States revealed that 25% of PWDs aged 18–65 years expressed hesitancy to receive COVID-19 vaccination.6 This hesitancy was associated with female sex, race, and education regarding the vaccine. In Bangladesh, PWDs had good knowledge, a positive attitude, and good behavioral practices in battling the COVID-19 pandemic.7 The majority of the PWDs followed health advisories during their in-patient and out-patient rehabilitation, hence it is assumed that they had access to information regarding the COVID-19 vaccination. Furthermore, the existing significant barriers for PWDs in Bangladesh to access healthcare, reliable information, long-term rehabilitation, social care, and support in livelihood8 were made worse by the COVID-19 pandemic. Challenges faced by PWDs during the pandemic included mobility issues, physical functioning, communication barriers, an inadequate workforce, and a lack of inclusive programs in health and rehabilitation in Bangladesh.9 There are a lack of relevant data in Southeast Asia on vaccine hesitancy or unwillingness for COVID-19 vaccination for persons with disabilities, but the study in the UK5, USA6, and Bangladeshi parents of neuro-developmental disability (NDD) children7 indicate there might be a significant number of PWDs in Bangladesh hesitant and unwilling to receive the COVID-19 vaccine. Besides, the parallel barriers to healthcare accessibility by PWDs in Bangladesh8,9 may contribute to the unwillingness or inaccessibility to receiving COVID-19 vaccines.\n\nHence the objectives of the study were to find out (1) the COVID-19 vaccination rate of persons with disabilities (PWDs) in Bangladesh, (2) the rate of unwillingness and inaccessibility to vaccination for them, and (3) predict the possible reasons for unwillingness or inaccessibility.\n\n\nMethods\n\nThis descriptive cross-sectional in-person survey of PWDs residing around the Centre for the Rehabilitation of the Paralysed (CRP) was carried out between February 2022 and May 2022. The Strengthening the Reporting of Observational Studies in Epidemiology (STROBE) guidelines for a cross-sectional survey were followed to conduct the study (Extended data 1).\n\nAny PWDs aged 12 years and more, with the ability to respond to the study questionnaire on their own or via a caregiver, was invited to participate in the study through an open call via social media, departmental notice and posters. A hospital-based randomization process was carried out from 12 service centers of CRP to enroll respondents in this in-person survey. The sample size has been calculated by Epi Info V 7.2.0 software (RRID:SCR_021682) from the Center for Disease Control (CDC) of the US, with a total PWD population of 4,480,000, and 25% the expected frequency, considering a 5% margin of error, and 1.0 design effect, a total of 288 samples could give a 95% confidence interval. We invited 396 persons with disabilities, and 241 PWDs responded to our invitation.\n\nA self-structured questionnaire was developed consisting of two parts: part I consisted of socio-demographic and clinical variables and part II contained of questions regarding acceptance of vaccination, knowledge, and attitude towards COVID-19 vaccination. The outcome variables were acceptance status by either positive answers, or negative answers to the screening question “Are you vaccinated?”. The indicator of the predictors was set as knowledge on COVID-19 vaccination, with 10 score questionnaires and three categorical questions on attitude towards COVID-19 vaccination. The knowledge score was calculated by 10 questions with three possible answers: “yes”, “no”, and “don’t know”. The questions were on “awareness about COVID-19 vaccine”, “benefits of vaccine for PWDs”, “importance in health”, “source of vaccine”, “dosage of vaccines”, “adverse effect of vaccines”, “immunity through vaccine”, “efficacy of vaccine”, and “health advisory after taking vaccines” (Extended data 2). The right answers scored 1, and wrong answers or “don’t know” scored 0. The total score was obtained by the summation of individual points that ranged between 0 and 10. The people with a total score 0–3 were categorized as having poor knowledge, 4–6 good knowledge, and 7–10 excellent knowledge.\n\nThe explanatory variables were socio-demographic information such as age, gender, marital status, residential area, education, occupation, family members at home, earning members, and monthly income. Health- and disability-related information such as type of disability, method of mobility, and comorbidity was also collected. The accessibility of vaccine was screened by another open-ended question “Were there any issues regarding the accessibility of vaccine for you?” This questionnaire was developed by a group of researchers from the microbiology, public health, and epidemiology background and re-examined by a national specialist of epidemiology.\n\nThe original questionnaire was developed in English and then translated into Bangla. Data were collected on the Bangla questionnaire by 15 trained data collectors who were fourth-year professional students of physiotherapy. Data entry and audit were performed by another group of students who were not involved in data collection but were trained on the questionnaire. The final dataset was screened by a statistician for consistency and data entry.\n\nBefore data collection, ethical approval from the Institutional Review Board (CRP/BHPI/IRB/02/2022/561) was obtained on February 1, 2022. Written informed consent was obtained from the respondents. Participants were assured about the confidentiality of data and their rights to withdraw their participation at any point in the study.\n\nData analysis was performed using IBM SPSS Statistics version 20.0 (RRID:SCR_019096) and Microsoft Excel 2016. Variables were determined as categorical or continuous data and considered either parametric or non-parametric based on data type, and normality tested through the Kolmogorov–Smirnov test and Shapiro–Wilk test. The descriptive statistics (Table 1) were reported as frequencies and percentages for the categorical data and measure of dispersions for interval and ratio data. Inferential statistics (Table 1) were carried out to determine the relationship between the outcome variable and predictor or explanatory variables using Chi-Square, independent t-test, or Fisher's exact test. Binary logistic regression (Table 2) was performed to find out the predictors of vaccine acceptance, considering vaccination status as a dependent variable and outcome or explanatory variables as predictor variables. The alpha level of significance was set at P< 0.05.\n\na Independent t test,\n\nb Chi-square test,\n\nc Fisher's exact test significant at\n\n* <.05,\n\n** <.01,\n\n*** <.001.\n\n* Significant with P<.05,\n\n** P<.01, and\n\n*** P<.001.\n\n\nResults\n\nA total of 241 persons with disabilities responded to our invitation, giving a response rate of 69%. A total of 186 (77.2%) PWDs had received one or more doses of COVID-19 vaccines of three doses supplied, and 55 (22.8%) were unwilling to go for a vaccination. Among the vaccine receivers, 186 (77.2%) received the first dose, 168 (69.7%) received both first and second doses, and six (2.5%) received all three doses, up until May 2022. Stroke survivors had the highest median days since receiving first and second doses (257, 192) than spinal cord injury survivors (170, 131) and others (169, 132). Figure 1 shows the median days of receiving shots among the vaccinated PWDs.\n\nAll (100%) of the persons with disabilities reported they were informed, invited, and aware of the vaccine campaigns near them, and the receivers (77.2%) stated vaccine camps were accessible to them. A total of 83% of the vaccinated individuals received the vaccine with the support of their family members, 10% were supported by a disabled people’s organization and 7% went alone to receive the vaccine. A total of 48% of the vaccinated people stated there was a separate booth for PWDs, and others said they were given special considerations.\n\nThe study included 151 spinal cord injury patients (62.7%), stroke survivors (22.4%), amputation and artificial limb users (10.8%) and others (4.1%). Other disabilities included adult cerebral palsy (n=3), Guillain-Barré syndrome (n=2), congenital myopathy (n= 2), congenital spinal deformity (n=2), and blindness (n=1). The respondents’ average age was 41.21±13.358 years, and the vaccinated PWDs mean age was slightly higher than the unwilling PWDs. The highest respondents were age 31–50 years (49.4%), followed by age 12–30 years (27.8%). A total of 22.8% of the respondents were elderly, aged more than 51 years. Males were the major respondents (64.7%) compared with females (35.3%). The majority were married (80.4%), belonged to the rural and semi-urban areas (63.4%), and 85.5% of respondents had a monthly family income of more than BDT 10,000. Hypertension was the major co-morbid condition (38.9%), followed by diabetes (26.9%), lung disease (22.4%), genito-urinary problems (24.6%), and heart disease (7.8%). Six respondents had a history of being COVID positive with the real-time polymerase chain reaction (RT-PCR) test before the vaccination. A total of 67.2% of the respondents were wheelchair users, 11.2% used a crutch, and 21.2% could walk with supervision (Table 1).\n\nParticipants were from diverse academic backgrounds: 4.1% had postgraduate degrees, 21.2% had graduation degrees, 22.8% had a higher secondary education, and 14.9% were illiterate. However, they had a mean score in vaccine-related knowledge of 5.97±2.314 in 0 to 10 scores. In addition, 16.6% had poor knowledge of vaccines (0–3 score), and 47.3% had excellent knowledge (7–10 score). A total of 89.2% had a positive attitude toward COVID-19 vaccination, but only 41.1% believed that the vaccine could benefit persons with disabilities (Table 1).\n\nA significant relationship (p<.05) was found between socio-demographic variables and vaccine status for age (P<.05), education (p<.01), area of origin (p<.01), disability status (p<.001), and mobility status (p<.001) (Table 1). However, the knowledge score and knowledge category regarding the COVID vaccine were also related to the status of the vaccine (p<.05). Figure 2 shows that the median knowledge score made a difference in vaccine acceptance or unwillingness to accept the vaccine. Attitudes towards COVID vaccine and belief also were related to vaccine acceptance or rejection (p<.05).\n\nTable 2 shows the predictors of unwillingness for COVID-19 vaccination. Spinal cord injury as a disability category was found to be a predictor of unwillingness to receive the vaccine (OR .36; 95% CI .19, .66; p<.01). PWDs from rural areas was also a predictor (OR .44; 95% CI .24, .81; p<.01). Other predictors were poor knowledge (OR .78; 95% CI .68, .89; p<.01), and dependency in mobility (OR.24; 95% CI.12, .45; p<.001).\n\n\nDiscussion\n\nA 77.2% acceptance rate and 22.8% unwillingness rate towards COVID-19 vaccines for PWDs in Bangladesh was seen. All participants had high accessibility of vaccine-related invitations, information, and booth availability for the PWDs. The vaccination booth operators were also cooperative and accessible to the PWDs. The predictive factors of unwillingness were spinal cord injury type of disability, people coming to the rehabilitation center from rural areas, poor knowledge of COVID-19 vaccination, and dependency on mobility.\n\nThe Centre for the Rehabilitation of the Paralysed (CRP) is the only comprehensive rehabilitation center providing both in-patient and out-patient specialized rehabilitation for spinal cord injury in Bangladesh. In 2021, the center served 279 spinal cord injury in-patients for a median length of 131 days, more than 3000 musculoskeletal and neurological cases, including stroke and amputee in-patients, for a median of 21 days, more than 1000 child in-patients with a disability for a median 14 days, and covered nearly 50000 out-patient PWDs through its 12 clinical service centers. As the population was drawn from the CRP, the study had respondents with spinal cord injury as the majority (62.7%), followed by stroke, amputees, and other forms of physical disability (Table 1). The age category was eligible from 12 years, keeping the ongoing vaccine campaigns (12 years and above) as of February 202210, but the majority of respondents were youths and young adults aged between 18 and 80 years. Males were the major respondents, and only six people tested COVID positive; the profile matches CRP’s patient demographics as there was only 300 RT-PCR tests performed at CRP in the year 2021. As the majority were spinal cord injury and stroke patients, the main method of mobility (67.2%) was either wheelchairs or assistive walking devices. A study on spinal cord injury at CRP11 suggests that the tetraplegia and paraplegia ratio of spinal cord injury was 0.7: 1; 67% of the discharged spinal cord injury patients used a wheelchair for mobility, with 85% of them originating from rural areas of Bangladesh. All the predictive factors except knowledge were associated with the disease burden or mobility issues of spinal cord injury (Table 2).\n\nOne study3 examined the unwillingness of receiving COVID-19 vaccination in Bangladesh for the general population. The study found a 27.4% unwilling rate, close to our study (22.8%). The study indicated that youths and young adults (18–25 years), education category, residence category, and non-adherence to the COVID-19 test were associated with unwillingness. Our study found a significant relationship between vaccine status and age category, education, and area of origin. Another study on the parents of NDD children in Bangladesh4 had similar association of vaccine hesitancy in age groups and areas of origin, but the study concluded that parents without an RT-PCR test or vaccination were significantly hesitant about receiving a vaccine for their children with disabilities. Studies on PWDs in the USA6 associated vaccine hesitancy with young age, demographic factors, and education; they also had factors associated with types of disability and the female sex. Our study could not find any relationship between vaccination status and sex category, but it found a relationship between vaccination status and disability and mobility status (Table 2). Our study findings are related to the existing national data of Bangladeshi people6,7 and the PWDs data of the USA6. The novelty of our study was to examine the “knowledge of the COVID-19 vaccine” as a predictive factor.\n\nWe examined the knowledge of COVID-19 vaccination with 10 questionnaire items prepared following the previously published studies12,13, with categorical answers “yes”, “no” or “don’t know”. The right answers scored as 1, wrong answers or “don’t know” were scored as 0. Our study had a mean score in vaccine-related knowledge of 5.97±2.314, as in category 16.6% had poor knowledge of vaccine (0–3 score), and 47.3% had excellent knowledge (7–10 score). Knowledge score was related to vaccination status (p<.05) (Table 1), and median scores were significantly different for PWDs with acceptance or unwillingness (Figure 2). The previous study suggests the level of knowledge of vaccines and sources of knowledge were factors that were indicative of the willingness to accept the vaccine.12 In addition, beliefs about the COVID-19 vaccine are another factor in the Southeast Asia region.13 We found 89.2% had a positive attitude toward COVID-19 vaccination, but only 41.1% believed that the vaccine could benefit PWDs. In regression analysis, we found poor knowledge of the COVID vaccine to be predictive of the unwillingness to be vaccinated; but we did not find any associations with attitude or belief. For persons with disabilities, accessibility and availability of the COVID-19 vaccine might be a factor in receiving the vaccine, but we found accessible information about the vaccine among the respondents. The respondents were invited and aware of the vaccine campaigns near them, and the camps were accessible to them.\n\nA total of 83% of the vaccinated individuals received the vaccine with the support of their family members, 10% were supported by a disabled people’s organization, and 7% went alone to receive the vaccine shot. In addition, 48% of the vaccinated people stated there was a separate booth for PWDs, while others said they were given special considerations. One study reveals14 that vaccination unwillingness could be predicted by the likelihood of risk, susceptibility, and severity of the disease. The respondents of our study had stable medical conditions with a variety of co-morbidities such as hypertension 38.9%, diabetes (26.9%), lung disease (22.4%), genito-urinary problems (24.6%), and heart disease (7.8%); none of these were linked to unwillingness or acceptance except hypertension (p<.05). Matched with knowledge level and rural origin, the unwilling respondents might have had no concern about their risk estimation of being out of the vaccination campaign; however, this was not evidenced by statistical calculation except support in mobility issues.\n\nThe strength of this unique study was to determine the predictive factors of the unwillingness for COVID-19 vaccination in persons with disabilities, who are vulnerable to the sequelae of COVID-19. Besides, the study examined two more important factors alongside the previous studies: knowledge, and accessibility to vaccines. The study had some limitations: respondents were clustered in an organization that was related to the rehabilitation of physical disabilities, hence other forms of disability were not accurately reflected in the study. We had a satisfactory response rate (69%) but were unable to fulfil the targeted sample size. Future studies adding the spectrum of disabilities, a larger sample size, and examining the impact of vaccination with post-COVID sequelae are recommended.\n\n\nConclusion\n\nThe study found that more than one-fifth of the persons with disabilities aged between 12 and 80 years were unwilling to receive the COVID-19 vaccination despite the accessibility of information and availability of the COVID-19 vaccine. The unwillingness was predictive of knowledge level, disability category, demography of origin, and mobility issues.\n\n\nEthical approval\n\nEthical approval was obtained from the Institutional Review Board (CRP/BHPI/IRB/02/2022/561) on February 1, 2022.\n\n\nConsent\n\nWritten informed consent was obtained from the respondents. Participants were assured about the confidentiality of data and their rights to withdraw their participation at any point in the study.\n\n\nAuthor contributions\n\nMZH, MAH, KMAH, VR contributed to conceptualization, data curation, formal analysis, funding acquisition, investigation, methodology, project administration, supervision, validation, visualization, writing—original draft preparation, writing—review and editing; MYAA, MHR, RA, SA, SJ, IKJ contributed to data curation, funding acquisition, methodology, validation, visualization, writing—original draft preparation, writing—review and editing.",
"appendix": "Data availability\n\nMendeley: Unwillingness of vaccine for PWD. https://doi.org/10.17632/4jmwvb7yt6.3. 15\n\nThe project contains the following underlying data:\n\n- Vaccine SPSS.sav\n\n- Vaccine.xlsx\n\nMendeley: Unwillingness of vaccine for PWD. https://doi.org/10.17632/4jmwvb7yt6.2. 15\n\nThis project contains the following extended data:\n\n- Questionnaire (in English).\n\nMendeley: STROBE checklist for Predictors of unwillingness or inaccessibility to receive the COVID-19 vaccination among persons with disabilities in Bangladesh. https://doi.org/10.17632/4jmwvb7yt6.3. 15\n\nMendeley: SAGER checklist for Predictors of unwillingness or inaccessibility to receive the COVID-19 vaccination among persons with disabilities in Bangladesh. https://doi.org/10.17632/4jmwvb7yt6.3. 15\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).\n\n\nAcknowledgments\n\nAuthors acknowledge the volunteers of Bangladesh Health Professions Institute (BHPI) for the data collection and volunteers of Amran’s School of Thoughts for their contribution to data audit.\n\n\nReferences\n\nRosen S: Covid-19 boosters and third doses. Coronavirus. 2021 [cited 2022 Nov 9].Reference Source\n\nAli M, Hossain A: What is the extent of COVID-19 vaccine hesitancy in Bangladesh? A cross-sectional rapid national survey. BMJ Open. 2021 Aug 1; 11(8): e050303. PubMed Abstract | Publisher Full Text | Free Full Text\n\nAli M: What is driving unwillingness to receive the COVID-19 vaccine in adult Bangladeshi after one year of vaccine rollout? Analysis of observational data. IJID Regions. 2022 Jun 1; 3: 177–182. PubMed Abstract | Publisher Full Text | Free Full Text\n\nAli M, Proma TS, Tasnim Z, et al.: Parental COVID-19 vaccine hesitancy for children with neurodevelopmental disorders: a cross-sectional survey. Tropical Medicine and Health. 2022 Dec; 50(1): 24–29. PubMed Abstract | Publisher Full Text | Free Full Text\n\nPaul E, Fancourt D: Predictors of uncertainty and unwillingness to receive the COVID-19 booster vaccine: An observational study of 22,139 fully vaccinated adults in the UK. The Lancet Regional Health-Europe. 2022 Mar 1; 14: 100317. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMyers A, Ipsen C, Lissau A: COVID-19 vaccination hesitancy among Americans with disabilities aged 18-65: An exploratory analysis. Disabil. Health J. 2022 Jan 1; 15(1): 101223. PubMed Abstract | Publisher Full Text | Free Full Text\n\nHossain MA, Hossain KA, Sakel M, et al.: Knowledge, attitudes, behavioural practises, and psychological impact relating to COVID-19 among people living with spinal cord injury during in-patient rehabilitation in Bangladesh. Front. Neurol. 2021; 12. Publisher Full Text\n\nHasan MT, Das AS, Ahmed AI, et al.: COVID-19 in Bangladesh: an especially difficult time for an invisible population. Disability & Society. 2021 Sep 14; 36(8): 1362–1367. Publisher Full Text\n\nKibria G, Islam T, Miah S, et al.: Barriers to healthcare services for persons with disabilities in Bangladesh amid the COVID-19 pandemic. Public Health in Practice. 2020 Nov; 1: 100027. PubMed Abstract | Publisher Full Text | Free Full Text\n\nCorrespondent S: Schoolchildren aged 12-17: Vaccination begins today at 8 centres. The Daily Star;2021 [Cited 2022 Nov 9].Reference Source\n\nHossain MS, Rahman MA, Herbert RD, et al.: Two-year survival following discharge from hospital after spinal cord injury in Bangladesh. Spinal Cord. 2016 Feb; 54(2): 132–136. PubMed Abstract | Publisher Full Text\n\nAl-Marshoudi S, Al-Balushi H, Al-Wahaibi A, et al.: Knowledge, Attitudes, and Practices (KAP) toward the COVID-19 vaccine in Oman: a pre-campaign cross-sectional study. Vaccines. 2021 Jun 4; 9(6): 602. PubMed Abstract | Publisher Full Text | Free Full Text\n\nNaeem S, Fraz TR, Zaid R, et al.: Understanding of COVID-19 vaccine knowledge, attitude, acceptance, and determinates of covid-19 vaccine acceptance among adult population in Pakistan. Journal of Xi'an Shiyou University (Natural Science Edition). 2022; 65(4). Publisher Full Text\n\nBrewer NT, Chapman GB, Gibbons FX, et al.: Meta-analysis of the relationship between risk perception and health behavior: the example of vaccination. Health Psychol. 2007 Mar; 26(2): 136–145. PubMed Abstract | Publisher Full Text\n\nZahid Hossain MDZ: Predictors of unwillingness or inaccessibility to receive the COVID-19 vaccination among persons with disabilities in Bangladesh. Mendeley. Publisher Full Text"
}
|
[
{
"id": "208315",
"date": "13 Oct 2023",
"name": "Sara Rotenberg",
"expertise": [
"Reviewer Expertise Health and disability",
"accessibility of vaccination"
],
"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 predictors of unwillingness suggest there is a perceived or real accessibility issue by individuals who haven't been vaccinated. However, the conclusion suggests that these are not valid factors. I think this needs to be reframed not about unwilling, but not being vaccinated or vaccine hesitancy. The term \"unwilling\" has negative connotations that don't capture the nuance of the results. There needs to be more discussion of this nuance in the results for the interpretation to be sound.\n\nIn the abstract, the conclusion makes it seem like everyone thought the vaccination centre was accessible to them, but only 77.2% agreed with that statement. It would be good to clarify this. Also, how did you verify the accessibility of vaccination sites?\n\nIt seems only people with physical impairments were included in the study. It would be good to clarify that that is the population, not all people with disabilities.\n\nIt would be important to compare these vaccination rates to people without physical impairments or national data to further contextualize the results.\n\nMedian time since vaccination is probably not meaningful now, since the data are over a year old.\n\n\"Descriptive status of respondents\" would be best moved to the beginning of the results section.\n\nFigure 2 does not have a legend to explain the results.\n\nIs the odds ratio adjusted or unadjusted?\n\nIn the discussion, it would be good to speak more about how vaccination could be improved for people with physical impairments to close this gap. Right now, the discussion repeats a lot of the results.\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Partly\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nPartly\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? No",
"responses": []
},
{
"id": "226233",
"date": "31 Jan 2024",
"name": "Kara B. Ayers",
"expertise": [
"Reviewer Expertise Disability",
"accessibility",
"Covid-19",
"inclusion",
"ableism",
"discrimination",
"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\nBased on my review, I have several suggestions to improve the rigor and clarity of this article on predictors of COVID-19 vaccine unwillingness among people with disabilities in Bangladesh: Main issues:\nThe article focuses solely on people with physical/mobility impairments, not all people with disabilities. This should be made clear, as experiences likely differ across disability types. Comparisons to vaccination rates in the general population would provide helpful context. Conflates unwillingness with lack of vaccination; the discussion should explore this nuance more given predictors like rural location, mobility dependencies, etc. which likely reflect barriers beyond just attitude. Conclusions stated in abstract don't fully align with results (e.g. on accessibility).\nOther suggestions:\nProvide more detail on survey accessibility considerations and any limitations. Move respondent descriptive data to start of results section. Add legend for Figure 2. Clarify if odds ratios are adjusted or unadjusted. In discussion, speak more to implications, e.g. how vaccination could be improved for this group to address disparities.\nOverall, I would recommend major revisions to improve clarity, nuance, and contextualization within disability and vaccination literature. But this is an important topic and with revisions could make a useful contribution.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Partly\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Partly",
"responses": []
}
] | 1
|
https://f1000research.com/articles/11-1564
|
https://f1000research.com/articles/11-1553/v1
|
21 Dec 22
|
{
"type": "Research Article",
"title": "Bottlenecks and opportunities towards achieving the targeted 95-95-95 HIV services in a rural district in Eastern Uganda",
"authors": [
"Monkya Samuel Namenkere",
"Ayaa Mary Stella",
"Sukuku Linda",
"Kharono Juliet",
"Mugabi Charles",
"Chelangat Benina",
"Mary Abwola Olwedo",
"Carol Nabasumba",
"Paul Oboth",
"Julius Osele",
"Rebecca Nekaka",
"Jacob Stanley Iramiot",
"Monkya Samuel Namenkere",
"Ayaa Mary Stella",
"Sukuku Linda",
"Kharono Juliet",
"Mugabi Charles",
"Chelangat Benina",
"Mary Abwola Olwedo",
"Carol Nabasumba",
"Paul Oboth",
"Julius Osele",
"Rebecca Nekaka"
],
"abstract": "Background: Uganda has made progress in reducing its HIV prevalence from 7.3% in 2011 to 6% in 2017, however, more needs to be done to meet the World Health Organization (WHO) target of 95% of the population knowing their HIV status, 95% enrolled on treatment and 95% achieving viral suppression. This study aimed to assess the bottlenecks and opportunities towards achieving the 95 95 95 targeted HIV services in the Bukedea district. Methods: A mixed-methods cross-sectional study was conducted in the Bukedea district covering males and females aged 18-65 years who had consented to participate in the study. We used a purposive sampling procedure to select our study participants. Qualitative data was collected through focus group discussions, key informant interviews, and document reviews for quantitative data. Quantitative data were analyzed using STATA v 14 whereas qualitative data were analyzed using the thematic analysis approach.\n\nResults: The challenges were grouped as patient-related, medication-related, and facility-related. The patient-related challenges were stigma, fear of taking the medication, poor nutrition, long distances, alcoholism, busy working schedules, and domestic violence. The medication-related challenges were side effects and pill burden. The facility-related challenges were inadequate pretest counseling and stock-outs. The use of anti-retroviral drugs (ART) was common in piggery and poultry and the sources of these drugs were reported to be the people on ART and the health workers.\n\nThe opportunities included home-based counseling, organizing more outreaches, counseling and health education, targeted testing, and strengthening the Village Health Teams (VHT) networks. Conclusions: The study revealed that the major challenges towards achieving the targeted 95-95-95 HIV services were stigma, inadequate pre-test counseling, fear of disclosure, and poor adherence due to alcoholism, sharing of drugs with animals and partners. The use of anti-retroviral drugs in animal husbandry was common in the Bukedea District.",
"keywords": [
"enrolment to care",
"HIV testing",
"Viral load suppression",
"HIV infection",
"HAART",
"expert clients",
"HIV care",
"Adherence to HIV treatment."
],
"content": "Introduction\n\nThere was a global decline in the number of new HIV infections from 3.4 million in 2001 to 2.3 million, indicating a 33% decline which has brought new hope in the fight against HIV/AIDS.1 The reductions in new HIV infection has been greatly observed among newborns, decreasing from 530,000 to 260,000 in 2000 and 2013 respectively due to national and global efforts invested in prevention of mother-to-child transmission (PMTCT) programs.1 Even with this progress, the number of HIV cases is steadily increasing with sub-Saharan Africa being the greatly affected region.2 Despite the high prevalence of HIV/AIDS in Africa, the Highly Active Anti-retroviral Therapy (HAART) coverage is still low.2 In Africa, an estimated 66% of eligible people living with HIV were not receiving HAART in 2013,3 only 45% of adults living with HIV were reported to know their HIV status, 86% of diagnosed persons were initiated on HAART, and an estimated 76% of persons on ART had achieved HIV viral load suppression in 2013.4 The Uganda Population-based HIV Impact Assessment (UPHIA) results revealed that the country has made significant progress in reducing the HIV prevalence from 7.3% in 2011 to 6% in 2017. There are 1.3 million people living with HIV in Uganda of which 73% know their HIV-positive status, 67% are on HAART, and 60% have achieved viral suppression.5 The World Health Organization set an ambitious 90-90-90 target6 which has now been upgraded to the 95, 95, 95 target; meaning 95% of the people living with HIV should know their status, 95% of diagnosed people should be enrolled into care, and 95% of those on ART achieve viral load suppression. In response, the Uganda Ministry of Health also set a 95, 95, 95 target. The progress of Uganda towards these targets is still worryingly low. Cases of low testing, poor adherence, and poor viral load suppression were common in Bukedea district. Our study assessed the bottlenecks and opportunities towards the targeted 95, 95, 95 HIV services in the Bukedea district.\n\n\nMethods\n\nA cross-sectional study was conducted between June 2020 and May 2021 in the Bukedea district using mixed methods. The study involved reviewing the existing data from the district information system and in-depth interviews. The study population comprised of people living with HIV, the health care workers involved in HIV care and treatment and the members of the community of Bukedea District. The document review was conducted to collect the summary information on trends in HIV testing, enrolment to care and viral load suppression. In the health facility, the health care workers who were directly involved in the care and treatment of HIV were purposively selected for the key informant interviews using an exploratory qualitative research design. Semi structured face-to-face interviews with the key informants were used to collect the data. The members of the Village health team were involved in a focus group discussion and also guided the selection of the members of the community to be involved in the focus group discussions. Semi structured face-to-face interviews were used to collect the data during the focus groups.\n\nThe Bukedea district is in Eastern Uganda, and is mainly inhabited by the Iteso tribe. It has a population of 203,600 people according to 2014 Census data, of which 98,684 (48.5%) are males and 104,916 (51.5%) are females, 74,533 are aged 0-9 years, 53,164 between 10-19 years, 45,151 between 20-39 years, 20,367 between 40-59 and 10,385 60 and above years. The biggest population are those under 20 years of age. 96.7% of the population stays in rural areas and 3.3% in urban areas according to the National Population and Housing Census.7\n\nThe study was conducted in two settings. The first was health facilities in Bukedea district and the second was the communities around the health facilities.\n\nWe reviewed existing information on HIV testing, enrolment to care and viral suppression from the HIV registers for Bukedea Health Center IV and also analyzed information on the Bukedea district dashboard to draw the general picture of the district’s performance towards achieving the 95-95-95 WHO target.\n\nPurposive sampling was used to select the key informant interview participants. The participants were selected based on the knowledge they had about the uptake of HIV services at Bukedea Health center IV and the district as a whole. Members of the village health team (VHTs), local leaders, and health care providers were selected to participate in this study. The participants were approached face to face with the guidance of the member of the Village Health teams. The sample size for the focus group discussions and the key informant interviews was determined by saturation, where the number was obtained after reaching a point where no new information was being gathered.\n\nMales and females aged 15-65 years were selected for the study. The participants below 18 years were included in this study because they make a sizable population of individuals living with HIV and some of them were non adherent. The participants below 18 years provided ascent and their parents/guardians provided consent. For HIV testing, both HIV positive and negative people were selected. For HIV treatment and viral load suppression, only HIV positive people currently on ART were selected.\n\nThe documents reviewed included the HIV testing register, enrolment to HIV care register, the viral load testing register and the district dashboard to capture the information from all the HIV care centers in Bukedea district. To assess the prevalence of HIV viral load non-suppression, we reviewed the ART register and the HIV viral load non suppressed clients’ register and analyzed the Bukedea district dashboard. The information on viral load suppression, HIV testing, adherence to treatment and the participants demographic data were collected using a data abstraction tool. The data abstraction tool contained the variables of interest of the study.\n\nThe focus group discussions were guided by the moderator who introduced topics for discussion to the participants who were given specific codes for identification. A total of 7 focus group discussions each consisting of 6-12 participants were conducted in the communities in Bukedea district. All the focus group discussions and the key informant interviews were audio-taped and transcribed verbatim for analysis.\n\nThe focus groups discussions involved people in the same age brackets and men and women were grouped separately. The focus groups discussions were conducted with the VHTs and the community members to collect data about the bottlenecks and the opportunities towards achieving the 95-95-95 HIV service in the Bukedea district. The key questions asked during the focus group discussions in the communities included: 1. What do you think are the obstacles that hinder the uptake of HIV testing services at Bukedea Health Center IV? 2. What could be the problem for the newly diagnosed people with HIV not being enrolled into care? 3. What factors contribute to non-adherence to treatment in your community? 4. What do you think are some of the opportunities that can be used to improve the uptake of HIV services at Bukedea Health Center IV?\n\nSemi-structured face-to-face key informant interviews were also used to collect data from the health care workers and HIV clients. Key informants were carefully selected based on the knowledge they had about the uptake of HIV services in the community. The key informants included the health workers of the health facility for example the ART in charge, the counselor, linkage facilitator, HIV viral load suppressed and non-suppressed clients in Bukedea district. The key questions asked during the key informant interviews included; 1. What could be the problem for the newly diagnosed people with HIV not being enrolled into care?, 2. What challenges do you face when providing HIV services to the people at your health facility?, 3. What factors contribute to non-adherence to treatment in among HIV patients?, 4. What do you think are some of the opportunities that can be used to improve the uptake of HIV services at Bukedea Health Center IV?\n\nThis was part and parcel of the research process right from data collection to data analysis. During the focus group discussion, the moderators ensured there was equal input all the participants. The separation of the focus group participants by age group and gender minimized the influence of the power dynamics on the information collected.\n\nThe tools used for the focus group discussions and key informant interviews were pre-tested before the actual data collection to ensure data quality. Member checking was also done to ensure that accurate information was collected.\n\nThematic data analysis was used. After data collection, the data was transcribed verbatim and analyzed using NVIVO v12. Two transcribers transcribed the data; the first transcriber transcribed the data and the second transcriber read the transcripts and listened to the audios to ensure the quality of transcription. Member checking was also done to ensure that there was no information loss on transcription. Two independent coders coded the data and the codes were discussed with a wider multi-disciplinary team of 12 members. After coding, all the codes and data extracts were collated for the later stages of data analysis. Themes were identified from the coded data which involved examining the codes and collated data to identify significant broader patterns of meaning. The themes were then reviewed by checking the candidate themes to ensure that a convincing story of the data was obtained and to ensure that the data was in line with the research objectives. The themes were then named and defined by a detailed analysis of each, working out the scope and focus of each theme. The final phase of data analysis involved writing the final report. The researchers then presented the findings and interpretations of the data.\n\nEthical clearance was sought from the Busitema University Faculty of Health Sciences Institutional Research Board, BUFHSREC02319. Confidentiality of the respondents was ensured and numbers were used for identification during the focus group discussions. Informed consent was obtained from the respondents and this was evidenced by a signature or thumbprint on the key informant guide. The participants were told that the information obtained from them would be reported in an anonymous form and published.\n\n\nResults\n\nA total of 90 participants took part in the study. Of these, 48 were community members, 12 were VHTs, 3 were health care providers which included the ART in charge, linkage facilitator, and the counselor, 12 participants were viral load non suppressed patients and 15 were viral load suppressed patients. 7 focus group discussion sessions and 30 face-to-face key informant interviews were conducted. The full transcripts for this study can be found under Underlying data.18\n\nA total of 24 community members that participated in the study were females and 24 were males, 32 of the community members were aged 20-65 years, 16 of them were aged between 18 and 20 years. The clients that participated in the study were those who had been enrolled in HIV care, 12 of them were HIV viral load suppressed clients and 15 were HIV viral load non-suppressed clients. 3 health care providers; the ART in charge, counselor, and linkage facilitator were interviewed. 12 VHTs participated in the focus group discussions.8 The duration of the key informant interviews was between 11 and 30 minutes with an average time of 24 minutes whereas the focus group interviews ranged between 50 minutes and 80 minutes.\n\nThe bottlenecks that emerged from the focus group discussions with community members and VHTs were classified as the client-related and facility-related factors. The client-related bottlenecks included; long distances to the health facility offering HIV care services, fear of disclosing their results to partners, stigma, and busy working schedules. The facility-related bottlenecks cited by the participants were bad experiences at the facility.\n\nStigma was commonly cited in the focus group discussions with the adolescents in the community. The stigma was mainly due to fear of their school peers knowing they are positive and also fear of starting medication:\n\n“HIV is a greatly feared disease in our community. People do not want to test for it and those who are tested and found positive refuse to be started on medication. I think the major cause of that is fear for their colleagues knowing about it and also fear to take the drugs.” (FG01)\n\nThe community members frequently mentioned that the fear of disclosing their positive HIV results to their spouses made them avoid testing for HIV. This was common among the females who feared intimidation from their husbands if they were found to be positive.\n\n“Here in Kachabule, a husband badly beat up his wife because she tested positive without informing him, on informing the husband about the results, he just blamed the lady, beat her up, and chased her away. This makes the wives fear to test.” (FG01)\n\nSome community members confessed that the busy working schedules they had, made them not turn up for testing because they work from the morning until the evening, as illustrated below.\n\n“I’m a carpenter and I do my work from morning to evening to get money for survival at home. I hardly get the time to go to the health center for testing and yet the health center is even far. Any time I miss not working is money being missed.” (FG02)\n\nSome people in the community did not test because of the long distances to the health facility. When asked about community outreach programs, they said that they were not aware of any outreach programs that take place in their villages.\n\nThe Uganda health system is organized in a hierarchical manner according to the population they serve. The National Referral Hospital serves a population of 30,000,000, the Regional Referral Hospital serves 2,000,000, the General hospital serves 500,000, the Health Center IV serves 70,000, the Health Center III serves 20,000, the Health Center II serves 5,000 and the Health Center I (Village Health Team) serves 1,000.9\n\nThe focus group discussions cited deterrent costs which they incur when the health facilities lack gloves and other sundries. All focus group discussions cited this challenge.\n\n“There is a time I went to the health center when I was suffering from malaria, they told me to buy gloves because since they had got over. From that time, I have never gone back to the facility and also not yet tested.”(FG03)\n\nThe bottlenecks in enrolment to care (the second 95) were also patient-related and facility-related. The patient-related challenges were fear of disclosure to the partners, fear to take the drugs, failure to believe in the positive results. The health care providers frequently mentioned that some people who turn positive after testing refuse to get enrolled into care because of fear to disclose the information to their partners. Even the ones that start treatment often hide the drugs from their partners. Moreover, because HIV treatment is for life, some people fear starting it. The ART in-charge mentioned that, due to this fear, some patients often said that they need time to think about whether to begin taking the medication or not. Some people take more time to understand their HIV results and end up leaving the facility.\n\n“A patient came, he was tested positive, and the patient did not accept that the results were his. He decided to move around various facilities testing to confirm if the results were positive. But after, he came back after some time because he had some complications and had to be started on ART.”(KI04)\n\nThe only facility-related challenge was inadequate pre-test counseling. This was reported by the counselor. The ART in charge said that inadequate pre-test counseling was because there were many testing points and only one counselor was attached to the facility.\n\nThe Bukedea HCIV had 960 clients enrolled on ART at the time of this study.\n\nThere was a higher number of females attending the HIV clinic than males (Table 2). The HIV viral non suppression was higher among the adult population (26/65 years) and lower among children and young adults below 25 years of age.\n\nThe bottlenecks in HIV suppression were classified as patient-related, facility-related challenges, and medication-related challenges. The patient-related bottlenecks included stigma and discrimination, alcoholism, poor nutrition, sharing of drugs with animals and partners, gender-based violence, lack of family support, religious beliefs, multiple partners, and poor medication routine.\n\nPatients and the health care workers frequently reported that stigma and discrimination made the patients stop taking their medication. Such patients ended up abandoning the drugs as illustrated in the quote below.\n\n“I used to miss my pills because of the people I used to stay with. My boyfriend did not know about my status and I was worried about revealing the truth because I did not want us to break up. I worry about how they may perceive it if they get to know that I'm on pills.”(KI07)\n\nSome women with undisclosed HIV status would hide their medication in odd areas such as inside the grass in the grass thatched house where drugs sometimes get wet in the rain season for the fear of their husbands discovering them. Some of the female participants confessed keeping their medication under the cooking stones where their husbands are least expected to check and find them.\n\nAlcoholism also came out strongly as a driver of non-adherence to the medication and hence non-suppression. Alcohol consumption was common among both men and women and in many cases, children.\n\n“I’m an askari, sometimes I take beer with my colleagues the moment I receive my salary just to be happy with them.”(KI03)\n\nFrom the focus group discussions in the community, there were several mentions of people sharing drugs with animals. The ARVs were given to pigs as fatteners and to chicken as a remedy for avian viral infections. Sharing ARVs was also reported to be common among spouses. Some reasons given for sharing of ARVs were; busy schedules, long lines in the health facilities and that men were afraid of being seen collecting their medication. This was frequently mentioned in the focus group discussions in the community as quoted below:\n\n“Here in Kachabule, there was a man who used to give ARVs to the pigs to make them grow fat and to hens to treat coccidiosis and I know many couples that share drugs especially those on the same regimen.”(FG01)\n\nOne of the key informants (the counselor) mentioned that gender-based violence greatly contributed to non-adherence. The commonly affected were the women who were abused by their husbands which caused them stress.\n\n“Some wives abandon the drugs because of family conflicts with their husbands. Some who divorce leave the drugs behind. This makes them abandon their medication.”(KI05)\n\nSome patients confessed that they missed medications simply due to forgetting to pick them up or not taking them on time. The natures of work and alcohol consumption especially among the security personnel, also often referred to as “askaris” were reported to be the common cause of skipping medication and refill appointments.\n\n“With the nature of my work, I have to wake up very early in the morning and report to duty because I'm an askari, sometimes I’m deployed to work in the nights and this makes me miss some days especially when my phone is off to check for the time.”\n\nPatients reported that one of the limiting factors to taking the drugs was lack of food to take them with.\n\n“We have been in the dry season, there are days we could only afford one meal a day, and this makes it hard for me to take the drugs because I could not take them with nothing to eat yet the drugs are too strong.”\n\nSome of the people living with HIV (PLWH) reported that they did not have support from their family members and this greatly contributed to missing medication.\n\n“When I'm with my wife, I sometimes forget to take the medication and I eat poorly but when I'm taken to my sister's place I find no problem in taking the drugs and I feel better.”\n\nThe community members and the adherence counselor reported that some patients who were on ART abandoned it because their religious leaders told them that Jesus would heal them without medication.\n\nThe other medication-related challenges included the pill burden, side effects, and resistance.\n\n“I stopped taking the drugs because I was tired of waking up every early morning which was so inconveniencing.”\n\nAdverse effects for example nightmares, confusion, dizziness, vomiting, and rashes on the skin made some patients stop taking the medication as illustrated below from an interview with a non-suppressed client.\n\n“From 2013, I was not having any challenges till recently when I began getting side effects for example dizziness. The challenge that I think is bringing about this is the shortage of food. I sometimes have nothing to eat and when I swallow the drugs, I begin feeling dizzy.”\n\nOne of the key informants reported that there were a few cases of the regimen failure for the patients especially those on second-line treatment. Stock-outs of vacutainers, loss to follow-ups were also common occurrences at the health facility.\n\n“There was a time they had stock outs for vacutainers and this made viral load monitoring for the patients and CD4 counts hard at the facility. The option they had was to refer the patients to other health facilities to check for their viral loads.”\n\nThis graph highlights the previous WHO targets of 90-90-90. The performance of the Bukedea district was still below the WHO set previous targets (Figure 1). While the district HIV detection and enrolment rates were getting closer to the previous targets, the viral suppression rates were still much lower.\n\nFrom the focus group discussions and key informant interviews we had with the community members, patients, VHTs, and health workers, we were able to identify several opportunities that could be utilized to achieve the targeted 95-95-95 HIV services in the Bukedea district.\n\nCounseling was seen as one of the ways stigma would be reduced and one of the factors that would contribute to adherence to treatment. At the facility, pre-test counseling was not adequately given and this explains why some people feared starting medication. Health education about the importance of taking the medication, the ways of ensuring proper adherence to treatment, and sensitization on the dangers of alcohol consumption could be used as an opportunity to improve adherence to treatment.\n\nTargeted testing was also identified as one of the ways of ensuring that all people living with HIV get tested. The targeted testing was done through testing the people more at risk of contracting the virus (HIV) for example the commercial sex workers, the truck drivers, the private security personnel, police officers, prisoners, and substance abusers.\n\nAlthough Assisted Partner Notification (APN) was used, it was, unfortunately, limited by inadequate funding. Assisted Partner Notification ensures that all the sexual partners of a positive client are screened and tested for HIV.\n\nFamily tracking was seen as one of the ways of making sure all people living with HIV test and get to know their status (Figure 2). This involves knowing the HIV statuses of the family members of the positive client comes from because of the possibility that they could have been born with the virus.\n\nFrom our study, we found out that self-disclosure by the expert clients on ART would help reduce the fear for stigma and also make the PLWH believe that if all the instructions for taking the medication are followed, a person living with HIV can have better quality of life and viral load suppression. The expert client in this study refers to the experienced HAART clients who have achieved viral load suppression. The expert clients have been co-opted to the Ugandan health care system to help in the management of HIV, especially to champion the message of adherence to treatment.\n\nMany people in the community said that they had not tested due to fear of being seen at the health facility while testing. We therefore saw home to home testing as one of the opportunities to make sure that all the people living with HIV know their status.\n\nMany people were not adhering to treatment because of gender-based violence and lack of family support. Linkage to social support networks ensures that the affected people are supported emotionally and financially.\n\nVHTs played a major role in providing counseling, ensuring that the clients in their communities keep appointments and go for refills, and also picked up refills for those who were unable to due to fear or other challenges.\n\nOne of the reasons patients gave for missing medication was forgetfulness. Setting reminders on phones for the clients who have them was seen as a way for patients to manage their medication.\n\nOne of the reasons why some people feared to be enrolled into care and also abandoned their medication was fear of disclose their positive status to the partners. We saw couple testing (Figure 3) as one of the ways of reducing the challenge because, through adequate pretest counseling, the married partners can prepare for either accordant or discordant results.\n\n\nDiscussion\n\nOur study sought to identify the challenges and opportunities towards achieving the targeted 95-95-95 HIV services in the Bukedea district. The challenges were classified as client-related, facility-related, and medication-related challenges. The challenges people face in getting tested for HIV included stigma, fear of disclosure of results to partners, long distances to health facilities, and busy working schedules.\n\nStigma, fear of disclosure of results, and long distances were identified as the major challenges that people encounter in getting to know their HIV status. Stigma was majorly reported from adolescent focus group discussions and this was due to fear of their friends knowing about their positive HIV status and fear of the lifelong treatment. Fear of disclosure of results was majorly reported by married females who feared intimidation from their husbands and also losing their marriages. The study also found out that long distances to the facility contributed to the poor turn up for the HIV testing services as reported by community members in the focus group discussions. These findings are similar to the results from a study carried out in 19 Ugandan districts to assess the availability, access, and utilization of HIV services.10 Results from several studies also reveal stigma, long distances, and fear of disclosure of results to partners being the major challenges faced by people when getting tested for HIV.\n\nAn important new finding this study was busy work schedules are a major hindrance to HIV testing as many people reported that they were always busy throughout the day and hardly get time for HIV testing.\n\nFor enrollment into care, the challenges found were fear of taking medication, fear of disclosure of the results, failure to accept positive results, and inadequate pre-test counseling. Because ARVs are taken over one’s lifetime, many people who tested positive for HIV didn’t want to enroll in care due to the daily bill burden and preferred to be initiated to care much later. There was fear of social discrimination as revealed by a study carried out in Bangladesh, Indonesia, and Vietnam to assess facilitators and barriers to retention in HIV care.11 Most married women refused to be enrolled into care for fear that they will receive abuse from their husbands if the results came out positive and they go back home with ARVs.\n\nAn important new finding from this study is the inadequate pretest counseling at the health facilities. Adequate pretest counseling prepares one's mind to receive the results and the clients enroll into care with adequate information. The challenge was majorly reported by the community members in the focus group discussions. The ART in charge attributed this to the fact that the health facility has many testing points with only one counselor attached to the facility making adequate pretest counseling a challenge.\n\nFor HIV viral load suppression, the challenges were alcoholism, stigma, and discrimination, sharing drugs with animals and partners, gender-based violence, pill burden, side effects, and resistance. The study found that alcoholism greatly contributes to poor HIV viral load suppression as confessed by some of the clients during the key informant interviews. After consuming alcohol, these clients get drunk and forget to take their medication hence affecting their treatment adherence. Alcoholism also changes the eating habits of HIV clients, which is a factor that greatly contributes to poor adherence. Some clients attributed poor adherence to pill burden as they have many ARV tablets and for life. A similar study in Zimbabwe about the failure of viral load suppression among adolescents on ART in Zimbabwe found out that the factors that contributed to the failure of viral load suppression among the clients included alcoholism, poor adherence, smoking, non-disclosure of the HIV status, and having more than one sexual partner in the previous twelve months.12\n\nStigma and discrimination remain a problem that was greatly reported by most of the clients who even confessed to abandoning their medication because of the community's perception of them. Due to fear of stigma and social discrimination, the clients end up abandoning the treatment leaving them to non-adherence. A similar study conducted in rural Uganda and Kenya found out that the challenges to achieving viral load suppression among children and adults in rural Uganda and Kenya included stigma and structural barriers to care for example, long wait times, frequent visits, opportunity costs to work, and inconvenience; all of which degrade retention in care hence poor adherence to treatment thus resulting in low viral load suppression.13\n\nReligious beliefs also majorly contributed to non-adherence to treatment. This was common amongst the clients who receive healing prayers from their religious leaders that make them lose confidence in the medication, leading to viral load non-suppression. A similar study conducted in Uganda to assess HIV viral load failure among perfect adherent HIV-positive adolescents showed that the rates of viral load suppression were higher among the adolescents who were adherent. However, 71% of the adherent adolescents had failed viral load suppression. The challenge to achieving viral load suppression among the adolescents was low adherence for people with strong spiritual beliefs who tend to miss medication during fasting and after receiving healing prayers and long exposure to antiretroviral therapy.14\n\nAn important new finding in this study is the sharing of medication with animals and partners which was reported by some participants during the focus group discussions and key informant interviews. Some of the reasons they gave for giving the drugs to animals were to enhance fattening in pigs and prevent diseases in chickens. The clients who confessed sharing drugs with partners reported that the long distance to the facility made them share drugs so that they could both come for their refills at the same time.\n\nAnother important finding from this study was the gender-based violence reported by the HIV counselor at the facility and study participants in the focus group discussions as one of the causes of poor adherence to treatment and majorly affected female clients who undergo psychological torture and stress. This makes them abandon the medication hence poor adherence and viral load suppression.\n\nThe opportunities towards achieving the targeted HIV services included home-based testing, targeted testing family tracking, counseling and health education, assisted partner notification, setting reminders, couple testing, linkage to social support networks, self-disclosure, strengthening the VHT network, and demonstrations on effects of alcohol.\n\nRoutine testing and counseling by the health care providers increases awareness about the importance of HIV testing and also encourages the clients to adhere to the medication because they get to know the importance of taking the medicine. This finding is similar to one from a study conducted in Uganda, Tanzania, and South Africa to assess factors strengthening HIV testing access.15\n\nAnother important opportunity is strengthening the VHT network through training them to help disseminate information to the community members through meetings and home visits hence increasing awareness about HIV. This is in line with a study conducted in rural Uganda to assess the perceived HIV testing norms. This study also suggests strengthening increased awareness about HIV testing through radio messages, billboards, and short text messages.16\n\nSelf-disclosure of the HIV status by the expert clients especially during intensive adherence counseling sessions and health education talks to the HIV clients leads to a reduction of fear to take the drugs and also fear for social discrimination amongst the HIV clients. Self-disclosure is consistent with findings from a study conducted in Harare city Zimbabwe.12 The same study also revealed that social support networks give support to the clients for example affected by gender-based violence, food shortages, unemployment, and those with no family support.12\n\nHome-based testing was also frequently mentioned in focus group discussions as one of the opportunities to increase uptake services. This helps to address the challenge of fear of confidentiality and also long distances to the health facility. A similar study conducted in southwestern Uganda about expanding HIV linkage into care found out that training of community extension workers led to home-based HIV testing.17\n\nNew important opportunities from our study include couple testing, assisted partner notifications, and demonstrations about alcohol effects. Demonstrations for clients about the effects of alcohol helped reduce the issue of alcoholism. This was done by putting one set of ARVs in water and the other set in alcohol. The one in water immediately dissolves and the one in alcohol doesn't. This helped reduce alcoholism and increased proper adherence to medication. Couple testing ensures with adequate couple pretest counseling reduces the challenge of fear of disclosure of the results to the marriage partners and also prepares the couple for the HIV results as a couple. The other important opportunity from this study is Assisted Partner Notification. This involves following up with all the sexual partners of an individual who tested positive and ensuring that all of them are screened and tested for HIV.\n\n\nConclusion and recommendations\n\nThe study revealed that the major challenges towards achieving the targeted 95-95-95 HIV services were stigma, inadequate pre-test counseling, fear of disclosure, and poor adherence due to alcoholism, and sharing of drugs with animals and partners. Therefore, continuous sensitization about HIV and the importance of adherence to drugs, continuous and adequate counseling of the clients on ART, and close monitoring of their viral load could help to improve enrollment into care, adherence to HIV treatment, and HIV viral load suppression.\n\n\nAuthor contributions\n\nMSN, AMS, SL, KJ, MC and CB concieved the idea, participated in data collection and wrote the first draft of the manusript, CN, PO, JO, RN, and JS did data curation, supervised data collection and conducted critical reviews. All authors read and approved the final version to be published.",
"appendix": "Data availability\n\nOSF: Bottlenecks and Opportunities towards Achieving the Targeted 95-95-95 HIV Services in a Rural District in Eastern Uganda. https://doi.org/10.17605/OSF.IO/BGNZ7. 18\n\nThis project contains the following underlying data:\n\n- 95 95 95 transcript.zip\n\nThis project contains the following extended data:\n\n- INTERVIEW GUIDES.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\nWe are grateful to our research participants who volunteered information to us and the in-charge Bukedea HCIV for the support in the data collection process.\n\n\nReferences\n\nDiese M, Shrestha L, Pradhan B, et al.: Bottlenecks and opportunities for delivering integrated pediatric HIV services in Nepal. Curr. Opin. HIV AIDS 2016; 11 Suppl 1(Suppl 1): S21–S29. PubMed Abstract | Publisher Full Text\n\nTessema B, Biadglegne F, Mulu A, et al.: Magnitude and determinants of nonadherence and nonreadiness to highly active antiretroviral therapy among people living with HIV/AIDS in Northwest Ethiopia: a cross - sectional study. AIDS Res. Ther. 2010; 7(1): 2. PubMed Abstract | Publisher Full Text | Free Full Text\n\nBilinski A, Birru E, Peckarsky M, et al.: Distance to care, enrollment and loss to follow-up of HIV patients during decentralization of antiretroviral therapy in Neno District Malawi:A retrospective cohort study PLOS;2017.\n\nNovitsky V, Gaolathe T, Mmalane M, et al.: Lack of Virological Suppression Among Young HIV-Positive Adults in Botswana. J. Acquir. Immune Defic. Syndr. 2018; 78(5): 557–565. PubMed Abstract | Publisher Full Text | Free Full Text\n\nUganda Go: Uganda HIV/AIDS Country Progress Report July 2016-June 2017; Theme: Reaching men, girls and young women to reduce new HIV infections UNAIDS – Joint United Nations Programme on HIV/AIDS;2018.\n\nSidibé M, Loures L, Samb B: The UNAIDS 90–90–90 target: a clear choice for ending AIDS and for sustainable health and development. J. Int. AIDS Soc. 2016; 19(1): 21133. PubMed Abstract | Publisher Full Text | Free Full Text\n\nThe Uganda Bureau of Statistics: The National Population and Housing Census 2014-Main report Kampala-Uganda;2016.Reference Source\n\nIramiot JS: Bottlenecks and Opportunities towards Achieving the Targeted 95-95-95 HIV Services in a Rural District in Eastern Uganda.2022.\n\nAmelia A, Walter A, Emmanuel A, et al.: Awareness of Antimicrobial Resistance among Primary Health Care Workers in Buyende District, Rural Eastern Uganda. Microbiology Research Journal International. 2017; 22(5): 1–11. Publisher Full Text\n\nBajunirwe F, Tumwebaze F, Akakimpa D, et al.: Towards 90-90-90 Target: Factors Influencing Availability, Access, and Utilization of HIV Services—A Qualitative Study in 19 Ugandan Districts. Biomed. Res. Int. 2018; 2018: 9619684.\n\nKoirala S, Deuba K, Nampaisan O, et al.: Facilitators and barriers for retention in HIV care between testing and treatment in Asia-A study in Bangladesh, Indonesia, Lao, Nepal, Pakistan, Philippines and Vietnam. PLoS One. 2017; 12(5): e0176914. PubMed Abstract | Publisher Full Text | Free Full Text\n\nSithole Z, Mbizvo E, Chonzi P, et al.: Virological failure among adolescents on ART, Harare City, 2017- a case-control study. BMC Infect. Dis. 2018; 18(1): 469. PubMed Abstract | Publisher Full Text | Free Full Text\n\nKwarisiima D, Kamya MR, Owaraganise A, et al.: High rates of viral suppression in adults and children with high CD4+ counts using a streamlined ART delivery model in the SEARCH trial in rural Uganda and Kenya. J. Int. AIDS Soc. 2017; 20(Suppl 4): 21673. PubMed Abstract | Publisher Full Text | Free Full Text\n\nNatukunda J, Kirabira P, Ong KIC, et al.: Virologic failure in HIV-positive adolescents with perfect adherence in Uganda: a cross-sectional study. Tropical Medicine and Health. 2019; 47(1): 8. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMcNaghten AD, Schilsky Mneimneh A, Farirai T, et al.: Implementation and Operational Research: Strengthening HIV Test Access and Treatment Uptake Study (Project STATUS): A Randomized Trial of HIV Testing and Counseling Interventions. J. Acquir. Immune Defic. Syndr. 2015; 70(4): e140–e146. PubMed Abstract | Publisher Full Text | Free Full Text\n\nPerkins JM, Nyakato VN, Kakuhikire B, et al.: Actual versus perceived HIV testing norms, and personal HIV testing uptake: a cross-sectional, population-based study in rural Uganda. AIDS Behav. 2018; 22(2): 616–28. PubMed Abstract | Publisher Full Text | Free Full Text\n\nAsiimwe S, Ross JM, Arinaitwe A, et al.: Expanding HIV testing and linkage to care in southwestern Uganda with community health extension workers. J. Int. AIDS Soc. 2017; 20(Suppl 4): 21633. PubMed Abstract | Publisher Full Text | Free Full Text\n\nIramiot JS:Bottlenecks and Opportunities Towards Achieving the Targeted 95-95-95 HIV Services in a Rural District in Eastern Uganda. [Dataset]. OSF 11 Oct. 2022. Web."
}
|
[
{
"id": "209374",
"date": "27 Nov 2023",
"name": "Kombatende Sikombe",
"expertise": [
"Reviewer Expertise HIV treatment"
],
"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\nAbstract- HIV prevalence from 7.3% in 2011 to 6% in 2017. Please provide more up to date data for this\nIntroduction- 3.4 million in 2001 to 2.3m in what year\n\nRef 5 is old. Are you sure the most up to date report for Uganda will report 73, 67, 60\nFor Key informant interviews in methods. Why not provide a supplement of the guide rather than list the questions asked. Write this to say we explored XYZ\nYour methods section is written to reflect that you collected Viral Load and did HIV testing but this isn't the case right. Like you say collected using a data extraction tool but was this from paper charts or the electronic health records.\n\nTable 1 should be your patient characteristics\n\nHow the health system is organised should go in the background and don't discuss this in the results\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?\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": []
}
] | 1
|
https://f1000research.com/articles/11-1553
|
https://f1000research.com/articles/11-1552/v1
|
21 Dec 22
|
{
"type": "Research Article",
"title": "The effects of antiretroviral stockout on primary health care nurses in the Ethekwini and Ilembe districts, KwaZulu-Natal",
"authors": [
"Farhana Mahomed",
"Emelda Zandile Gumede",
"Emelda Zandile Gumede"
],
"abstract": "Background: Nurses face challenges of antiretroviral therapy (ART) stock out when rolling out, leading to drug resistance, poor compliance and decreased viral suppression. Poor viral suppression leads to higher morbidity and mortality rates, hence a need to strengthen supply chain principles in order to achieve equal distribution of resources amongst clinics. Nurses also need to have relevant guidelines available to prevent treatment failure. The study aimed to describe the effects of ART stockouts on primary health nurses rolling out ART in clinics in KwaZulu Natal. Methods: The study followed a qualitative approach, using a descriptive design. Two primary healthcare clinics in Ilembe and Ethekwini districts were selected for the study. The population comprised of 8 purposively selected participants. In-depth interviews and focus groups were conducted to describe the experiences of primary health care nurses rolling out antiretroviral therapy regarding stockout and how it was managed. Data collection and analysis followed Yates, Partridge and Bruce steps. Ethics and trustworthiness were maintained. Results: The singular theme of inconsistent availability of treatment emerged, as well as sub-themes of means used to manage treatment stockout and supply change management issues. Conclusions: An adequate supply of ART through an efficient supply chain management strategy, ongoing training in primary health care and nurse-initiated management of antiretroviral therapy and the availability of treatment guidelines should be enforced.",
"keywords": [
"antiretroviral therapy",
"primary health care",
"supply chain management"
],
"content": "Introduction\n\nStockouts are defined as the non-availability of a required medicine for at least a day at a storage or delivery point.1 Stock shortages cause nurses to either dispense a lower amount than what was prescribed or temporarily change antiretroviral (ARV) regimen.2 Suboptimal coordination between government donors and lack of human resources have been seen as causes for stock-outs.3 Pathways for sharing information between different levels in the Department of Health (DOH) about medical supply levels are not well-defined, especially when the tender contract ends and the new one commences.4 Sub-district pharmacists must be vested with such information, so as to prepare enough stock to cover the transition period, thus avoiding stock-out.\n\nWeaknesses in ARV supply chains and poor coordinated services frequently contribute to stockout.5 Due to this issue, nurses are to give a reduced supply of medication to patients, resulting in short return dates, and overburdened workloads as patients return more frequently to clinics to collect treatment that was initially insufficient. In these cases, clients leave the health facility with either an insufficient supply or no medication at all.6 Despite this practice of decanting medication so that a patient does not leave the facility without it, there is no legislation governing such practices.\n\nDue to ART stockout, sometimes primary health care nurses opt to issue an alternate drug as per the treatment guidelines, that is only where there is a possibility of fatal allergies. This is the predicament that is facing the nurses, and they wonder if their professional bodies are going to support such initiatives or not, hence, the researcher conducted this study which aimed to describe the effects of ART stockout on primary health care (PHC) nurses in the Ethekweni and Ilembe districts.\n\n\nMethods\n\nA qualitative research design with a specific focus on a descriptive design using the constructivist paradigm to describe the effects of ART stockout on primary health nurses initiating ART was used in this study. It focused on the qualitative aspects of their lived experience as they rollout ART and the challenges they face.7\n\nThe study was conducted for almost 6 weeks from 23rd August 2021 to 30th September 2021 in clinics in the Ethekweni and Ilembe District, respectively. Ilembe District is situated on the east coast of KwaZulu-Natal Province; it is the smallest of the province's districts and includes four health sub-districts, namely Mandeni, KwaDukuza, Maphumulo and Ndwedwe. The Ethekwini district is the economic centre of the Province, and consists of features of an industrialized society, a high disease burden, a large number of informal settlements and highly mobile populations. To ensure that no bias influenced the results, tape recordings and field notes were utilised and those were kept for further auditing.\n\nThe inclusion criteria were primary health nurses initiating ART working in ARV clinics with a minimum of 2 years of experience. The exclusion criteria were primary health nurses initiating ARVS, that did not have 2 years of experience working in ARV clinics.\n\nNon-probability purposive sampling was used to recruit participants for individual interviews and two focus group discussions (FGD). Individual in-depth interviews from a clinic in each district were conducted. The researcher wanted to recruit as many participants as possible, but not all met the inclusion criteria, hence managed to get 8. All 8 participants did the individual interviews and 7 participants were eligible for the FDG. The interview allowed the participants to express their experiences in detail. Probing was used to encourage participants to elaborate and provide a detailed exploration on their experiences working in primary healthcare clinics. Two open-ended questions were used to guide the interview, which included the following: What are the reasons for ART stock outs? and How is it managed?\n\nFollow-up questions in the form of probes and prompts were asked based on the participant’s response to the questions.\n\nThe focus group interview allowed the participants to share their thoughts with each other, producing new ideas and reflecting a range of views before answering.7 Because of coronavirus disease 2019 (COVID-19) outbreak, social distancing and its protocols were maintained throughout the focus group discussion.\n\nAfter obtaining permission from the relevant departments, the researcher engaged in the process of getting the relevant details for all participants that met the eligibility criteria and communicated the information and received consent from them, maintaining COVID-19 infection control protocols. The information sheet was in English only, as English is the language used by nurses in KwaZulu Natal. There was no need for an interpreter. Those nurses that had given consent to participate in the study, communicated with the researcher face to face, maintaining social distancing. Observations were done by the researcher twice at each clinic, for approximately 4 hours per day. This was done so that she could observe the behaviors of the participants and collect data by using field notes. Firstly, the researcher posed as a volunteer working in the clinic and carried out observations in their natural setting to avoid a Hawthorne effect. All data collection methods including these observations, were approved by the ethics committee (see end of manuscript for full details), and consent was obtained from the chief executive officer (CEOs/PHC) managers of the relevant institutions before any data was collected.\n\nSecondly, the researcher revealed her identity and observations and field notes were done in person as this enabled her to gain a picture on how the participants interacted with patients rolling out antiretroviral therapy.\n\nThe researcher holds a master’s degree in nursing, working in a PHC clinic with 8 years of experience. She also did research modules in ethics, in possession of a training and resources in research ethics evaluation (TRREE) certificate. The first encounter was with gatekeeper permission. Participants were only aware of the aim of the study, through the information sheet. The interviews took place in a quiet consultation room in each clinic and were audio recorded. The discussion began informally with the researcher greeting the participants and laying out the instructions for the interview process. The interview sessions for each participant lasted between 30-45 minutes. Each interview continued to the point where the participant could not provide any new information. Data saturation was reached after the 6th participant, however, two more interviews were held for validation purposes.\n\nData was transcribed verbatim using data analysis stages for the phenomenographic studies as described by Yates, Partridge and Bruce.8 These stages were used to uncover variation in how the phenomenon under study was perceived. Data analysis was done with thematic analysis which led to the emergence of themes and subthemes. All transcribed and translated data were transferred to the qualitative data analysis software package NVIVO 12.\n\nTrustworthiness and rigor were maintained throughout the study using the criteria of Lincoln and Guba.9 The focus group discussions allowed the participants to share their thoughts with each another, producing new ideas and reflecting on a range of views before responding to posed questions.7\n\nTo ensure credibility, the researcher personally collected, transcribed, and analyzed data to ensure prolonged engagement. The researcher carried out member checking through probing and paraphrasing participants’ responses during the interview process to ensure that the information captured was a true reflection of what the participants were saying. Confirmability was ensured with the use of audio recordings and field notes that were utilized and kept for further auditing.\n\nThe researcher transcribed the audio recordings verbatim. Dependability was achieved, as the researcher used an interview guide, based on the study objectives and research questions to ask the participants more or less similar questions. To ensure transferability, the researcher provided in-depth descriptions of information concerning the participants, their settings and the context.\n\nData was analyzed in stages using Yates, Partridge and Bruce’s format shown in Table 1.\n\nOnce the above stages were complete, the researcher developed themes of each transcript. The themes of individual transcripts were then compared for similarities and variation which led to the compilation of the theme and subthemes.\n\n\nResults\n\nThe participants that were selected for the study were trained in PHC with experience in nurse initiated management of antiretroviral therapy (NIMART). Their experience working in PHC clinics ranged from 2 and 13 years. Those that did not meet the eligibility criteria were excluded from the study. Four of the nurses from the clinic in the Ethekwini district were not trained in primary health care. Two of the nurses from the clinic in the Ilembe district did not have the minimum of 2 years experience to be part of the study. The theme that emerged was the inconsistent availability of treatment which merged with supply chain management issues and means to manage treatment stockout.14\n\nParticipants expressed their concerns that there was a shortage of treatment, and they could not function efficiently to meet patient’s needs. By not having sufficient medication, the participants extended their scope of practice, by decanting medication into bottles and supplying to patients. Participants stated that they were unsure about the correct stock levels at the clinic. Instead of patients receiving a full 3month supply of medication, they were issued with only a month supply.\n\nThe participants stated that during certain months, the department was showing over supply of medication and they were baffled with the reasons leading to this stock levels. They expressed concern over the functioning of the procurement system as they sometimes experienced over or under supply of ART, hence the concern is ART stockout.\n\nP2, Clinic A “The pharmacist will tell us that during certain months, we are showing over stockage and the next month is under. We are not sure how the procurement system functions within the district because we had a lot of ARV stock outs, and it's unexplainable”.\n\nParticipants mentioned that they have to decant medication into packets for issue to patients due to the limited stock. The issued medication would cover patients for a few days. Other participants stated that instead of giving a patient a full 3month supply of medication, they issued a month supply, as the stock was inadequate. This meant that patients incurred high financial costs due to travelling to the clinic more frequently. Patients were more prone to defaulting, drug resistance, non-compliance and higher morbidity and mortality rates.\n\nP4, Clinic A “We had to open up these bottles and decant the tablets into packets and give the patients just to cover for a few days. So, you do what you have to”.\n\nP3, Clinic B “We serve a number of ART patients on antiretrovirals and highly active antiretroviral therapy (HAART). Most of them are migrant workers that require more than one month's supply. Instead of a 3month supply, we only issue a month’s supply because we don’t have enough”.\n\n\nDiscussion\n\nParticipants mentioned that they had to devise ways to assist patients, even if it meant that the tablets lasted for a short while. There is no legislation governing such practices. If a patient becomes resistant to ART in future, the health system will be liable for such atrocities. PHC nurses do not have documentation or guidelines to follow, in the event of medication stockouts. PHC nurses are extending their scope of practice to assist patients, but this is not ethical, in terms of the duties they should be carrying out. Health-care workers are using refill periods, borrowing medicine, or referring patients to other facilities using coping strategies to provide ART to patients when there are stockouts in their facilities.10 The same sentiment is shared in a study conducted by.11 In ART facilities, ART dispensers were dispensing doses that ranged from 15 days to 3 months depending on the patient’s condition. Frequent stock-outs forced them to dispense medicines even for a single day.\n\nParticipants mentioned that they were unsure of how the procurement system worked as they had experienced many stockouts of ART at the clinic. PHC nurses should be educated by managers of clinics on how medication is procured and delivered to the clinic pharmacies. This will give the nurses an idea of how the supply chain system works. A 2015 study from Kinshasa found that stock-outs of HIV commodities were common especially due to supply chain problems, like late deliveries.12\n\nParticipants stated that there were discrepancies in their stock levels as they were told by the pharmacists that there were over supplies of ART issued to their clinics, where in fact, there were actual shortages in ART. In a state conference on 3rd June 2019 in Johannesburg by the Stop Stockouts, it was mentioned that a supplier responsible for delivery, delivered over 1 million additional packs of second line ARVs, when only 128,000 packs were ordered by National Department of Health, and the extra packets are nowhere to be found and no one is accountable for them.13 This study had many strengths. Participants shared their lived experiences as they rolled out ART. The study was conducted in their natural environment at their own time and it was done in their language so there was no interpreter needed. Participants expressed how their challenges ought to be managed. We only collected data from 1 clinic in each of the 2 districts, which cannot represent the heterogeneity of all PHC nurse’s experiences of clinics in the other districts.\n\n\nConclusion\n\nThis study concludes that there is a need to strengthen the stock systems in health institutions in KwaZulu Natal. Having an adequate supply of ART, will enhance the smooth running of clinics and give PHC nurses the chance to work more efficiently, hence providing quality care to patients. It is recommended that the district pharmacists work together with the Department of health and the companies who are awarded tenders to supply the ART, so that the pharmacist will know when the present tender is ending and the new one commencing, so that she/he may prepare adequate supply to cover the transition period.\n\nAn adequate supply of ART through an efficient supply chain management strategy should be made available. Close tracking of stock should be implemented when it is delivered to an institution and entered into the system that will monitor its movement, so that each stock is accounted for in time, and shortages are managed. As the levels drop, the supply process of the next stock will be underway, and the cycle of events run smoothly. It is also recommended that there should be ongoing training in primary health care and NIMART, so that the nurses will be sure of the correct drug replacement in cases of shortages, thus avoiding drug reactions leading to medical emergencies in cases of incorrect drug dispensation. Updated standard operating procedures (SOPs) and the availability of treatment guidelines should be implemented, so, in the event of ART stockouts, PHC nurses will be aware of who to contact in cases of such, as well as their acceptable scope of practice, regarding dispensing and decanting of available drugs amongst the patients in the clinic.\n\nThe ethical clearance was obtained on the 12th of August 2021 from the Biomedical Research ethics committee of UKZN (BREC00002821/2021). Permission was sought and obtained from the Provincial Research Ethics Committee. Consent was obtained from the chief executive officer (CEOs/PHC) managers of the relevant institutions before any data was collected. The names of the clinics were protected to ensure confidentiality. Written Informed consent was obtained from participants before the commencement of individual interviews and focus group interviews. The anonymity of the participants was ensured by assigning pseudo names and codes. Participation was voluntary and data safety was maintained throughout the study. All methods were performed in accordance with the relevant guidelines and regulations.",
"appendix": "Data availability\n\nFigshare: Antiretroviral stockout. https://doi.org/10.6084/m9.figshare.21485526. 14\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 are extremely grateful to the nurses from the clinics in the two districts for their valuable time and support during the study. An earlier version of this article can be found on Research Square (doi:01.21203/rs.3.rs-1970213/v1).\n\n\nReferences\n\nWorld Health Organization (WHO): National AIDS programmes: A guide to indicators for monitoring and evaluating national antiretroviral programmes. Geneva:2005.\n\nWorld Health Organization: Guideline on when to start antiretroviral therapy and on pre-exposure prophylaxis for HIV. Geneva:2015.\n\nSchouten EJ, Jahn A, Ben-Smith A, et al.: Antiretroviral drug supply challenges in the era of scaling up ART in Malawi. Journal of International AIDS Society. 2011; 14(suppl 1): S4.PubMed Abstract | Publisher Full Text | Free Full Text\n\nStop Stockouts: 4th National survey report. The fragile system.2017.\n\nMinior T, Douglas M, Edgil D, et al.: The Critical Role of Supply Chains in Preventing Human Immunodeficiency Virus Drug Resistance in Low- and Middle-Income Settings. J. Infect. Dis. 2017; 216(9): S812–S815. PubMed Abstract | Publisher Full Text | Free Full Text\n\nHwang B, Shroufi A, Gils T, et al.: Stock-outs of antiretroviral and tuberculosis medicines in South Africa: A national cross-sectional survey. PLoS One. 2019; 14(3): e0212405.PubMed Abstract | Publisher Full Text | Free Full Text\n\nBrink H, Van der Walt C, Van Rensburg G: Fundamentals of Research Methodology for Healthcare Professionals. Juta and Company;(4th Edition).2018.\n\nYates C, Partridge H, Bruce C: Exploring information experiences though phenomenography. Libr. Inf. Res. 2012; 36(112): 96–119.Publisher Full Text\n\nLincoln Y, Guba E: Naturalistic Enquiry. Beverly Hills, CA:SAGE Publications;1985.\n\nHodes R, Price L, Bungane N, et al.: How frontline healthcare workers respond to stockouts of essential medicines in the Eastern Cape Province of South Africa. S. Afr. Med. J. 2017; 107(9): 738–740.PubMed Abstract | Publisher Full Text\n\nBerhanemeskel E, Beedemariam G, Fenta T: HIV/AIDS related commodities supply chain management in public health facilities of Addis Ababa, Ethiopia: a cross-sectional survey. J. Pharm. Policy Pract. 2016; 9: 11.PubMed Abstract | Publisher Full Text | Free Full Text\n\nGils T, Bossard C, Verdonck K, et al.: Stockouts of HIV commodities in public health facilities in Kinshasha: Barriers to end HIV. PLoS One. Public Library of Science. 2018; 13(1): 1–12.\n\nThe Stop Stockouts Project: SSP alarmed at second line ARV and contraceptive shortages across South Africa.2019.\n\nMahomed F, Gumede EZ:Antiretroviral stockout. figshare. [Dataset].2022. Publisher Full Text"
}
|
[
{
"id": "158702",
"date": "16 Jan 2023",
"name": "Abosede Adekunbi Farotimi",
"expertise": [
"Reviewer Expertise Health Education and Community/Public Health Issues"
],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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: Please, note that the title is not appropriate. The word \" effects\" should be replaced with \"challenges \" it can be restated thus; Challenges of primary health care nurses regarding antiretroviral stock out in Ethekwini and Ilembe districts, Kwazulu-natal.\nINTRODUCTION: The introduction is scanty. The researchers need to cite the examples of other studies that are related to this study. Concepts like Nurse initiated management of antiretroviral therapy (NIMART) and change management issues should be addressed. Broad and specific objectives need to be clearly stated.\nLITERATURE REVIEW : the literature review is scanty.\nMETHODOLOGY: The reason(s) for choosing the design should be stated. The reason(s) for using purposive sampling should be explained. More information on the setting should be provided, for example, the capacity of the facility. There is need to explain the criteria for choosing the two districts under study. Please, note that only two open- ended questions cannot give details for in-depth interview.\nANALYSIS: In the analysis, the demographic profile of participants were not mentioned.\nDISCUSSION: Discussion needs to be revisited. It is very scanty.\nCONCLUSION: Conclusions are drawn fairly well\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Partly\n\nAre sufficient details of methods and analysis provided to allow replication by others? Partly\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nNot applicable\n\nAre all the source data underlying the results available to ensure full reproducibility? Partly\n\nAre the conclusions drawn adequately supported by the results? Partly",
"responses": []
},
{
"id": "250993",
"date": "10 Apr 2024",
"name": "Tsitsi B. Masvawure",
"expertise": [
"Reviewer Expertise HIV prevention and treatment. Medical anthropology. Global 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 paper examines the effects of ART stockout on healthcare providers in KwaZulu-Natal. The topic of medication stockouts in the context of the HIV pandemic is important as it has dire implications for medication resistance and viral suppression. It can also negatively affect the morale and motivation of healthcare personnel. However, despite the importance of the topic, this paper presents scant study findings and the entire paper is very brief. The three results presented are essentially that there are regular medication stockouts, the healthcare staff do not know why these occur and they respond to these stockouts by dispensing smaller quantities of drugs to patients. The paper is lacking the deep dive that characterizes qualitative research. We also learn very little about the effect of these stockouts on healthcare personnel, as suggested by the title of the paper. If the journal word count is the main constraint, the authors should consider truncating the methods section (which is very detailed) and devoting more space to presenting more study findings. The main weakness of the paper is lack of data.\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": []
}
] | 1
|
https://f1000research.com/articles/11-1552
|
https://f1000research.com/articles/11-289/v1
|
09 Mar 22
|
{
"type": "Research Article",
"title": "Anti-angiogenic effect of the combination low-dose sorafenib and EGCG in HCC-induced Wistar rats",
"authors": [
"Andry Irawan",
"Erik Prabowo",
"Ignatius Riwanto",
"Wahyuni Lukita Atmodjo",
"Erik Prabowo",
"Ignatius Riwanto",
"Wahyuni Lukita Atmodjo"
],
"abstract": "Background: Sorafenib is an expensive standard drug used for advanced hepatocellular carcinoma. Its combination with epigallo-3-catechin gallate leads to a reduced cost but equally effective anti-angiogenic effect. Therefore, this study aims to assess the anti-angiogenic effect of standard-dose Sorafenib compared to the combination of low-dose Sorafenib and epigallo-3-catechin gallate. Methods: A total of 25 male Wistar rats (7-weeks-old) were randomly divided into 4 groups, namely Sham (K), Control (O), combination of low-dose Sorafenib and epigallo-3-catechin gallate group (X1), and standard-dose Sorafenib group (X2). All groups were injected with N-Nitrosodiethylamine 70 mg/kg bodyweight (BW) intraperitoneally for 10 weeks, except the Sham group. After the development of hepatocellular carcinoma, X1 and X2 were treated for 2 weeks. Subsequently, the level of vascular endothelial growth factor (VEGF) and expression of microvascular density was examined using liver tissues. Results: There was a significant difference (p=0.007) in the level of VEGF between the group X1 (106,682 ± 41,024) and X2 (214,5162 ± 67,71652). However, the differences in VEGF level of group X1 and X2 compared to group O (318,101 ± 55,078) were significantly lower, with values p=0.000136 and p=0.019, respectively. The expression of microvascular density between groups X1 (36 ± 4,416) and X2 (26,2 ± 4,55) was not significantly different. Meanwhile, a significant difference (p<0.05) was discovered when both groups were compared with group O (176 ± 19). Conclusion: The combination of low-dose Sorafenib with epigallo-3-catechin gallate is superior in reducing the level of VEGF compared to standard-dose Sorafenib and is better than the control. Standard-dose Sorafenib as well as the combination of low-dose Sorafenib and epigallo-3-catechin gallate have similar effectivity to reduce the expression of microvascular density.",
"keywords": [
"Sorafenib",
"Epigallo-3-Catechin Gallate",
"Vascular Endothelial Growth Factor",
"Microvascular Density",
"N-Nitrosodiethylamine"
],
"content": "Introduction\n\nHepatocellular carcinoma (HCC) is the most common primary type of liver cancer. In 2013, the prevalence of liver and bile duct cancer in a developed country like the United States was 30,640.1,2 Meanwhile, a high incidence of HCC was discovered in South and East Asia, Central and West Africa, Melanesia, and Micronesia/Polynesia. It has been estimated that there are more than 749,000 new cases of HCC in men and 226,000 in women every year.3,4 In 2020, liver cancer was considered the sixth most common cancer and the third leading to cancer-related deaths in the world.5\n\nVascular endothelial growth factors (VEGF) play an essential role in HCC tumor growth. Several carcinogens and tumor promoters initiate inappropriate activation of nuclear factor kappa B (NF-kB), which mediates the inflammation process and tumorigenesis. Meanwhile, overexpression of VEGF increases blood vessels permeability, leading to the differences between oxygen flow and delivery. A high level of VEGF is also typical in chronic liver disease that often triggers HCC.6,7 Micro-vessel density (MVD) is a tumor indicator of angiogenesis that needs to be examined in HCC since a higher level of MVD shows a poor prognosis. This high angiogenic activity can be inhibited through the administration of anti-angiogenic drugs.8\n\nThe most common management of HCC for operable cancers is liver resection, while chemotherapy and targeted therapy are also used. It was discovered that 80% of HCC patients are diagnosed with advanced-stage or inoperable cancer. Systemic therapy with Sorafenib is required to change the condition at the operable stage. Sorafenib has been proven to be the first systemic therapy that successfully improved HCC patients’ survival rate. It is an oral multi-kinase inhibitor that targets vascular endothelial growth factor receptor (VEGFR)-1, VEGFR-2, and VEGFR-3, thereby, reducing tumor angiogenesis. The disadvantages of Sorafenib treatment include high cost and approximately 30% of all patients responded to the treatment. Monotherapy of Sorafenib can cause several patient complaints, resistance, and high costs, therefore, when given in low-dose and combination with herbal medicines, the same effect is expected which is more affordable in price.9–12\n\nEpigallocatechin-3-gallate (EGCG) is an active ingredient that was proven to prevent the growth of blood vessels in experimental animals. Its mechanism of action is by inhibiting urokinase and tyrosine kinase, which activates VEGF, epidermal growth factor (EGF), and fibroblast growth factor (FGF).13 In 2005, a previous study in Japanese stated that EGCG induces both in vitro and in vivo liver cell apoptosis to improve the prognosis of HCC. In vitro studies showed that the effective concentration of EGCG varies from 1 to 100 mol/L. According to pre-clinical studies that were carried out in rats, only less than 5% of oral catechin taken as a constituent of tea can reach systemic circulation, therefore, intraperitoneally administration is considered more effective. EGCG is the right choice to be combined with Sorafenib in advanced HCC, which uses the synergism of the two drugs. This combination can lead to similar effects as the Sorafenib standard dose.14,15\n\nTherefore, this study aims to investigate the effectiveness of anti-angiogenic activity between Sorafenib standard dose and the combination of low dosage Sorafenib with EGCG. It was presented in adherence to the checklist of ARRIVE reporting guidelines.\n\n\nMethods\n\nThis study used a randomized control trial post-test only design method (Figure 1). A total of 25 male Wistar rats (PT Biomedical Technology Indonesia), which were 7 weeks old with bodyweight 200–250 grams were placed in a cage with a controlled temperature of 22°C under 12 hours of light and dark cycle. The rats were given free access to food with AIN76 standard dietary formula for rodents, which was 67.7% carbohydrates, 11.5% lipids, and 20.8% protein from the Food Engineering Laboratory, IPB, Bogor, Indonesia, purchased from PT Surya Science and Beverages.\n\nDEN: Diethyl-Nitrosamine, EGCG: Epigallocatechin-3-gallate, MVD: Micro-vessel density, VEGF: Vascular endothelial growth factor.\n\nDiethyl-Nitrosamine (DEN) (N0756) with a molecular weight of 102.14 and Epigallocathecin-3-O-Gallate (Y0001936; primary pharmaceutical grade standard) with a molecular weight of 458.37 was purchased from Sigma-Aldrich. While, each tablet of Sorafenib contains 200 mg as Tosylate (Nexavar).\n\nThis study followed the National Institutes of Health Guidelines for the Care and Use of Laboratory Animals and was approved by Mochtar Riady Institute for Nanotechnology Ethics Committee (MRIN EC) with protocol number 2101001-AS06. The inclusion criteria were healthy and active 7-week-old male Wistar rats weighing 200–250 grams, while unhealthy male Wistar rats with anatomical anomalies are excluded. During the experiment, any infected or dead Wistar rats were also dropped out.\n\nThe sample size was calculated using the degree of freedom (Minimum and Maximum sample) formula. Rats were randomized and allocated into 4 groups, consisting of 7 rats in each group, except the control (K) group that contained 4 rats, with a minimal sample size of three. Subsequently, DEN was injected intraperitoneally in the abdominal area below the umbilicus on 21 rats for two treatment groups and a control group, with 70 mg/kg BW/week for 10 weeks.16,17 After 10 weeks, all rats were randomly divided into 4 groups, namely sham (K), Sorafenib 5 mg/kg BW + EGCG 5 mg/kg BW (X1), Sorafenib 15 mg/kg BW (X2), and without treatment (Group O). Group K was injected with saline for 10 weeks parallel with other groups. After the administration of DEN, group O was sacrificed, and a pathologist carried out a histopathological examination of liver tissue to show the formation of HCC. During the examination, anaplastic cells, oval nucleus, pleomorphic, coarse chromatin, and nucleolus invasive growth into stroma were observed, which confirmed HCC.\n\nSorafenib was dissolved in a maximum of 1.5 mL saline (maximum 10 mL/kg BW/day) and administered orally at a dose of 5 mg/kg BW and 15 mg/kg BW. Subsequently, EGCG 5 mg/kg BW/day was dissolved in approximately 1.5 mL saline (maximum 20 mL/kg BW/day) and administered by intraperitoneal injection once a day for 14 days. The sham group (K) was administered a saline solution orally and intraperitoneally, while the sorafenib-only group (X2) received intraperitoneal saline and oral Sorafenib. Meanwhile, the combination group of EGCG and Sorafenib (X1) received intraperitoneal EGCG and oral Sorafenib and the bodyweight of the rats was measured once a week. At the end of the experiment, the rats were sacrificed, exsanguination was done on deeply anesthetized animals with ketamine 80 mg/kg BW and xylazine 100 mg/kg BW intramuscularly to ameliorate any suffering, and the liver tissues were resected and examined microscopically. Moreover, a veterinarian carried out a necropsy when any rat died during the experiment to investigate the cause of death investigate the cause of death (Figure 2).\n\nGroup X1, X2, and O present multiple white nodules (White arrow); group (K) did not develop any nodules.\n\nIn liver tissue, VEGF was evaluated using an enzyme-linked immunosorbent assay (ELISA) quantitative methods, while MVD was calculated using Immunohistochemistry (IHC). The intensity and area of sinusoidal endothelial staining were measured quantitatively using a microscope at 100× magnification. Furthermore, the hot spots from the immunohistochemistry were selected using the “color selection” function and the “area/density (intensity)” function (ImageJ, RRID:SCR_003070) to calculate the values.\n\nTo prepare lysate from tissue, tissue of interest was dissected with clean tools. Dissected tissue was placed in microcentrifuge or Eppendorf tubes. Lysis buffer consisting of NP-40 buffer, sodium chloride, NP-40, Tris pH 8.0, and Triton X-100 or NP-40) was added to 5 mg of tissue and homogenized rapidly. Next, it was centrifuged at 4°C for 20 minutes. After carefully removing the tubes and placing them on ice, any supernatant was aspirated, and the pellet were discarded.18,19\n\nA Bradford, a Lowry, or a bicinchoninic acid (BCA) assay was conducted to calculate protein concentration. Bovine Serum Albumin (BSA) is usually used as a standard protein. Each sample was frozen at -20°C for immunoprecipitation. 200μL of 1X Bradford reagent, 5 μL of BSA, and 30 μL of the unknown sample were added to each test tube. Absorbance was determined using sipper or individual cuvettes at 595 (VIS lamp).\n\nAll standards and samples were prepared twice as recommended and stored at room temperature. Each well containing standard and sample were incubated for 2.5 hours at room temperature. The washing process using 300 μL of Wash Buffer was repeated four times. All liquid was eliminated after each step to achieve the best result. After the last wash, the plate was inverted and blotted using a paper towel.\n\nApproximately 100 μL of 1× Detection Antibody was titrated and incubated at room temperature for one hour. All liquid was removed, 100 μL of Streptavidin solution was added and incubated at room temperature for 45 minutes. Approximately 100 μL of TMB One-Step Substrate Reagent (Item H) was added and incubated in dark condition for 30 minutes. Lastly, 50 μL of Stop Solution (Item I) was added and absorbance was recorded at 450 nm.\n\nDeparaffinization was done in incubator at 60°C for 45 minutes, followed by deparaffinization in xylene for 10 minutes. Next, 96% ethanol, 80% ethanol, and 70% ethanol were subsequently added to the formalin-fixed paraffin-embedded tissue for 5 minutes. Tissue was washed using distilled water. Antigen retrieval buffer (citrate buffer + tween) was placed into a jar and microwaved in full power for 20 minutes. The jar was removed and chilled on ice for 20 minutes.\n\nSeveral drops of Hydrogen Peroxide Block were added to the section, incubated for 10 minutes, and rinsed two times in buffer. Protein Block was then added, incubated for 10 minutes, and rinsed once in buffer. Primary MVD polyclonal antibody (MBS 2520154) was added (1:100) in PBS-T, incubated in 4°C for 2 hours, and rinsed four times in buffer. A Biotinylated Goat Anti-Polyvalent was added, incubated for 10 minutes, and rinsed four times in buffer. Streptavidin Peroxidase was added, incubated for 10 minutes, and rinsed four times in buffer.\n\nApproximately 30 μL of DAB Chromogen was applied into 1.5 mL of DAB Substrate. It was incubated for 3 seconds and rinsed four times in buffer. Next, Hematoxylin was used as a counterstain, incubated for 20 minutes, and rinsed in tap water. Tissues were dehydrated using 70% ethanol, 80% ethanol, and 96% ethanol, each for 1 minute. The samples were observed under 10×, 40×, and 100× magnification.20 For MVD, the Spearman’s correlation coefficient (rho) was 0.93 (p < 0.01), while intra-observer agreement (Kappa) were 0.88 for cut-off using mean.20,21\n\nAll data were expressed as mean ± standard deviation of the mean. The statistical analysis was carried out using SPSS 28 (IBM SPSS Statistics, RRID:SCR_019096). All data were normally distributed and the comparisons between groups were analyzed using ANOVA. Post hoc analysis using the least significant difference (LSD), where a p-value < 0.05 was considered statistically significant.\n\n\nResults\n\nThis study showed that the Sorafenib-only group effectively reduced VEGF tissue level better than without the treatment group. However, there was no significant difference in reducing MVD expression compared to the combination of low-dose Sorafenib and EGCG group, which indicated better overall results than the Sorafenib-only group. During the experiment, 9 rats died due to pulmonary hemorrhage and one rat died with irregular lung surface.\n\nA total of 13 rats survived the 11 weeks of the experiment, although some looked unhealthy. One group reached the minimum sample size based on the calculation of the degree of freedom, the Institutional Animal Care and Use Committee (IACUC) Guidebook, and the World Health Organization (WHO). At the end of the experiment, the whole group of mice was terminated according to the euthanasia techniques based on IACUC and the American Veterinary Medical Association (AVMA) Guidelines.\n\nThe Shapiro-Wilk test results were used to calculate the mean and the distribution of the rats’ body weight data and a p > 0.05 was obtained for all groups. Homogeneity test results with Levene’s test obtained p = 0.978, which showed that the data obtained is homogeneous.\n\nFrom the 16 sample slides examined, it was discovered that the tumor growth was solid with anaplastic cells having round, oval, pleomorphic nuclei, coarse chromatin, and prominent nucleolus that grows invasively into the stroma (Figure 3).\n\nMicroscopic 40× magnification on non-induced Diethyl-Nitrosamine (DEN) group: (A, Group K) normal hepatocyte; comparing graphic on DEN induced group: (B, Group O) Bile Duct hyperplasia (Black square area); (C, Group X1) Hyperchromatic cells (black-arrow) and (D, Group X2) Prominent cell (thin-arrow), Hyperchromatic cell (thick black – arrow), Ballooning degeneration (Blue-Arrow). HT, Hepatocyte; PT, Porta tract.\n\nThe mean VEGF values (Figure 4) between group Sorafenib 5 mg/kg BW + EGCG 5 mg/kg BW (X1) (106,682 ± 41,024) and group Sorafenib 15 mg/kg BW (X2) (214,5162 ± 67,717) had significant difference (p < 0.05), which showed that group X1 had the strongest effect in reducing VEGF values. Furthermore, the VEGF values between group X1 and the group without treatment (O) (318,101 ± 55,078) was significantly different (p < 0.05). A similar result was seen since VEGF values between group X2 and O was significantly different (p < 0.05).\n\nThere was significant difference in levels of VEGF in group X1 compared to X2 (*). Between group X1 and O, VEGF was significantly different (**). Between group X2 and O, VEGF was significantly different (***) (p < 0.05).\n\nMVD evaluation was carried out using 10× and 40× magnification to measure the intensity and area of sinusoidal endothelial staining (Figure 5). Subsequently, the hot spots from the immunohistochemistry were selected, and values were calculated. The mean MVD expression between the group Sorafenib 5 mg/kg BW + EGCG 5 mg/kg BW (X1) (36 ± 4.416) and group Sorafenib 15 mg/kg BW (X2) (26.2 ± 4.55) had no significant difference. Based on the results, MVD expression (Figure 6) between all groups and group O (176 ± 19) showed a significant difference (p < 0.05).\n\nAll three groups showed a “hot spot” area, which means there were positive results.\n\nThere was no significant difference in expression of MVD between group X1 and X2, but both X1 and X2 were significantly different compared to the control group (O) (non-treatment group) with (p < 0.05).\n\n\nDiscussion\n\nThis study showed that the benefits of the combination of Sorafenib and EGCG are the same as an anti-neoplastic drug, which is as effective as anti-angiogenic. The results of VEGF values between 2 groups, namely the combination group Sorafenib 5 mg/kg BW and EGCG 5 mg/kg BW (X1) compared with Sorafenib 15 mg/kg BW alone (X2) showed that both treatments can reduce VEGF values. However, the X1 group was significantly more potent in decreasing VEGF value compared to group X2. This has exceeded the expectations, where the combination of low-dose Sorafenib and EGCG was more effective than only standard-dose Sorafenib. This indicated that Sorafenib and EGCG act synergistically with strengthening effect in anti-angiogenesis.\n\nIn vivo and in vitro studies have proved the effect of EGCG as a chemo-preventive, anti-angiogenic, anti-invasive, anti-proliferative, anti-inflammatory, and antioxidant substance. It was shown that EGCG blocks NF-kB activation by inhibiting IκBα degradation and the mitogen-activated protein kinase (MAPK) pathway. Meanwhile, downregulation of inducible nitric oxide synthase (iNOS) transcription and nitric oxide (NO) production from macrophages is dependent on NF-kB inhibition. It was reported that EGCG blocks NF-kB activation in human endothelial cells and inhibits monocyte chemotactic protein-1 (MCP-1) expression. Similarly, EGCG also prevents the apoptosis process by reducing mRNA expression of Bax and caspase 3 activity. It also inhibits cyclooxygenase-2 (COX-2) expression, proteasome-dependent degradation, MAPK pathways, and growth factor-dependent signaling, namely insulin-like growth factor-I (IGF-I), VEGF, and EGF.22\n\nThe results also suggested that several factors are responsible for the less effectiveness of the administration of Sorafenib as a single drug. Firstly, Sorafenib is accumulated in cancer cells, followed by an increase in the expression of enzymes to metabolize Sorafenib, which affects drug exposure. Thirdly, the presence or absence of tumor influences the concentration of Sorafenib and its primary metabolites based on assessing resistance to Sorafenib administration.23\n\nMVD expression was significantly different between X1 and X2 groups compared to group O. This showed that the combination of low-dose Sorafenib and EGCG is also effective as standard-dose Sorafenib-only by decreasing MVD expression intratumorally. However, there is no significant difference in the discovery of MVD expression between the X1 and X2 groups. These are influenced by time length because the formation of MVD is affected by the growth of the capsule in the tumor. This is in line with the results by Kuczynski EA et al., who showed that therapy with Sorafenib significantly inhibits MVD (p < 0.001 vs. controls), while the Sorafenib-resistant group showed no evidence of continued angiogenesis.24 The evaluation of MVD expression is critical to determine the prognosis. According to Poon RTP et al., MVD-CD34 tumors were the only significant predictor of disease-free survival in patients with HCC or tumor size < 5 cm.25\n\nAlthough the result was different from the hypothesis, a very satisfying conclusion was successfully obtained. The combination of low-dose Sorafenib with EGCG had better effectiveness than the standard-dose of Sorafenib in lowering VEGF values and was equally effective in reducing MVD expression in Wistar rats induced by DEN. Based on the results, it was concluded that EGCG adds a supplementary anti-angiogenic effect for HCC. Therefore, the use of low-dose Sorafenib combined with EGCG acts as a more cost-effective therapy is recommended to potentially increase drug compliance. Similarly, it also provides a satisfying therapeutic effect for advanced HCC.\n\nAfter 10 weeks of administering DEN 70 mg/kg, macroscopic gross liver tissues showed irregular surfaces and pale colors, the histopathologists also confirmed HCC characteristics. The length and dosage of DEN induction were in line with Atmodjo et al., while the liver carcinogenesis or the beginning of HCC was recorded.26 There was a force majeure event in the experimental animal since 9 rats were found dead. Meanwhile, 8 rats died because of pulmonary hemorrhage, and one died with irregular lung surface due to lung injury. The hypothesis of this study stated that the suppression of the immune system increases the probability of lung disorders due to respiratory infections or fibrosis, or early malignancy in the lungs when rats were injected with DEN.27 This is because one rat that was not administered DEN died due to lung hemorrhage. This can be explained by Kun MW et al., who discovered the other cause of Wistar rat’s lung problem was lung infection due to A.Cantonensis.28 M. Pulmonis causes a different type of infection as explained by the study of Chawla et al., which showed gross and histopathological discoveries of severe congestion of lungs with suppurative and necrotizing pneumonia.29 Wang Y et al. also evaluated the induction effect of DEN in a rat model and discovered that the induced rat had liver dysfunction and damage, which is characterized by diffuse lesions with extensive interstitial inflammatory cell infiltration, alveolar edema, and bleeding. Meanwhile, minor injuries were discovered in the spleen, kidney, large intestine, heart, and other organs.30 Atmodjo et al. also noted the same discoveries for lung hemorrhage.26\n\nThere are several limitations to this study, firstly, the unhealthy condition of the samples after 10 weeks of administration of DEN can affect the number of samples. This led to the consideration of the decision of earlier termination. Secondly, the method of administering EGCG was unclear with the best effectiveness, oral EGCG which is associated with poor absorption (<5% absorption rate). Therefore, the intraperitoneal injection method was used to administer EGCG. This study recommends further investigation whether the administration of EGCG in form of nanoparticles orally or parenterally can increase the absorption and bioavailability of EGCG in the intestine and plasma.12,13\n\n\nConclusion\n\nThe combination of low-dose Sorafenib with EGCG has a more potential anti-angiogenic effect in liver cancer by reducing VEGF values compared to the single standard-dose Sorafenib. It also has similar effectiveness as single standard-dose Sorafenib in reducing MVD expression compared to the control group. Meanwhile, further study on the anti-angiogenic effect of low-dose sorafenib combined with EGCG is recommended.\n\n\nData availability\n\nZenodo: Underlying data for ‘Anti-angiogenic effect of the combination low-dose sorafenib and EGCG in HCC-induced Wistar rats.’ https://doi.org/10.5281/zenodo.6044890.\n\nThis project contains the following underlying data:\n\n- VEGF and MVD raw data.xlsx (dataset)\n\nZenodo: ARRIVE checklist for ‘Anti-angiogenic effect of the combination low-dose sorafenib and EGCG in HCC-induced Wistar rats.’ https://doi.org/10.5281/zenodo.6044890.\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\nAuthors’ contributions\n\nConception and design: Andry Irawan, Erik Prabowo, Ignatius Riwanto\n\nAdministrative support: Andry Irawan, Erik Prabowo, Wahyuni Lukita Atmodjo\n\nProvision of study materials or patients: Wahyuni Lukita Atmodjo\n\nCollection and assembly of data: Andry Irawan\n\nData analysis and interpretation: Ignatius Riwanto, Wahyuni Lukita Atmodjo\n\nManuscript writing: All authors\n\nFinal approval of manuscript: All authors",
"appendix": "Acknowledgements\n\nWe thank Pelita Harapan University for facilitating this study and Dr. Ricarhdo Valentino Hanafi for organizing the manuscript.\n\n\nReferences\n\nCenters for Disease Control and Prevention (CDC): Hepatocellular carcinoma - United States, 2001-2006. MMWR Morb. Mortal. Wkly. Rep. 2010; 59(17): 517–520.\n\nCrissien AM, Frenette C: Current management of hepatocellular carcinoma. Gastroenterol. Hepatol. (N Y). 2014; 10(3): 153–161. PubMed Abstract\n\nFerlay J, Shin HR, Bray F, et al.: Estimates of worldwide burden of cancer in 2008: GLOBOCAN 2008. Int. J. Cancer. 2010; 127(12): 2893–2917. PubMed Abstract | Publisher Full Text\n\nShimizu M, Shirakami Y, Sakai H, et al.: Chemopreventive potential of green tea catechins in hepatocellular carcinoma. Int. J. Mol. Sci. 2015; 16(3): 6124–6139. PubMed Abstract | Publisher Full Text\n\nInternational Agency for Research on Cancer: Liver. World Health Organization; 2020. Accessed: January 30, 2021.Reference Source\n\nJain RK, Tong RT, Munn LL: Effect of vascular normalization by antiangiogenic therapy on interstitial hypertension, peritumor edema, and lymphatic metastasis: insights from a mathematical model. Cancer Res. 2007; 67(6): 2729–2735. PubMed Abstract | Publisher Full Text\n\nAmarapurkar AD, Amarapurkar DN, Vibhav S, et al.: Angiogenesis in chronic liver disease. Ann. Hepatol. 2007; 6: 170–173. Publisher Full Text\n\nBösmüller H, Pfefferle V, Bittar Z, et al.: Microvessel density and angiogenesis in primary hepatic malignancies: Differential expression of CD31 and VEGFR-2 in hepatocellular carcinoma and intrahepatic cholangiocarcinoma. Pathol. Res. Pract. 2018; 214(8): 1136–1141. PubMed Abstract | Publisher Full Text\n\nSun HC, Tang ZY: Angiogenesis in hepatocellular carcinoma: the retrospectives and perspectives. J. Cancer Res. Clin. Oncol. 2004; 130: 307–319. PubMed Abstract | Publisher Full Text\n\nLlovet JM, Ricci S, Mazzaferro V: Sorefenib in advanced hepatocellular carcinoma. N. Engl. J. Med. 2008; 359(4): 378–390. Publisher Full Text\n\nMacGregor JL, Silvers DN, Grossman ME, et al.: Sorafenib-induced erythema multiforme. J. Am. Acad. Dermatol. 2007; 56: 527–528. PubMed Abstract | Publisher Full Text\n\nCao Y, Cao R: Angiogenesis inhibited by drinking tea. Nature. 1999; 398(6726): 381. PubMed Abstract | Publisher Full Text\n\nFassina G, Vene R, Morini M, et al.: Mechanism of inhibition of tumor angiogenesis and vascular tumor growth by epigallocatechin 3 gallate. Clin. Cancer Res. 2004; 10: 4865–4873. PubMed Abstract | Publisher Full Text\n\nLi Y, Chang SC, Goldstein BY, et al.: Green tea consumption, inflammation and the risk of primary hepatocellular carcinoma in a Chinese population. Cancer Epidemiol. 2011; 35: 362–368. PubMed Abstract | Publisher Full Text\n\nNishikawa T, Nakajima T, Moriguchi M, et al.: A green tea polyphenol, epigalocatechin-3-gallate, induces apoptosis of human hepatocellular carcinoma, possibly through inhibition of Bcl-2 family proteins. J. Hepatol. 2006; 44(6): 1074–1082. PubMed Abstract | Publisher Full Text\n\nNishida H, Omori M, Fukutomi Y, et al.: Inhibitory effects of (−)-epigallocatechin gallate on spontaneous hepatoma in C3H/HeNCrj mice and human hepatoma-derived PLC/PRF/5 cells. Jpn. J. Cancer Res. 1994; 85: 221–225. PubMed Abstract | Publisher Full Text | Free Full Text\n\nArifin WN, Zahiruddin WM: Sample Size Calculation in Animal Studies Using Resource Equation Approach. Malays. J. Med. Sci. 2017; 24(5): 101–105. PubMed Abstract | Publisher Full Text\n\nMathonnet M, Descottes B, Valleix D, et al.: VEGF in hepatocellular carcinoma and surrounding cirrhotic liver tissues. World J. Gastroenterol. 2006; 12(5): 830–831. PubMed Abstract | Publisher Full Text | Free Full Text\n\nLandriscina M, Cassano A, Ratto C, et al.: Quantitative analysis of basic fibroblast growth factor and vascular endothelial growth factor in human colorectal cancer. Br. J. Cancer. 1998; 78(6): 765–770. PubMed Abstract | Publisher Full Text | Free Full Text\n\nViera AJ, Garrett JM: Understanding interobserver agreement: the kappa statistic. Fam. Med. 2005; 37(5): 360–363. PubMed Abstract\n\nAgnani B, Solanki R, Ansari M, et al.: Prognostic Significance of Microvessel Density as Assessed by anti CD34 Monoclonal Antibody in Invasive Ductal Carcinoma of Breast. Asian Pac. J. Cancer Prev. 2021; 5(3): 75–79.\n\nJantan I, Ahmad W, Bukhari SNA: Plant-derived immunomodulators: an insight on their preclinical evaluation and clinical trials. Front. Plant Sci. 2015; 6: 1–18.\n\nRochat B: Role of cytochrome P450 activity in the fate of anticancer agents and in drug resistance: focus on tamoxifen, paclitaxel and imatinib metabolism. Clin. Parmacokint. 2005; 44: 349–366. PubMed Abstract | Publisher Full Text\n\nKuczynski EA, Lee CR, Man S, et al.: Effects of Sorafenib Dose on Acquired Reversible Resistance and Toxicity in Hepatocellular Carcinoma. Cancer Res. 2015; 75: 2510–2519. PubMed Abstract | Publisher Full Text\n\nPoon RT, Ng IO, Lau C, et al.: Tumour microvessel density as a predictor of recurrence after resection of hepatocellular carcinoma: a prospective study. J. Clin. Oncol. 2002; 20: 1775–1785. PubMed Abstract | Publisher Full Text\n\nAtmodjo WL, Larasati YO, Isbandiati D, et al.: Curcuminoids Suppress the Number of Transformed-Hepatocytes and Ki67 Expression in Mice Liver Carcinogenesis Induced by Diethylnitrosamine. J. Can. Sci. Res. 2018; 3: 2.\n\nBurkholder T, Foltz C, Karlsson E, et al.: Health Evaluation of Experimental Laboratory Mice. Curr. Protoc. Mouse. Biol. 2012; 2: 145–165. PubMed Abstract | Publisher Full Text\n\nWun MK, Davies S, Spielman D, et al.: Gross, microscopic, radiologic, echocardiographic and haematological findings in rats experimentally infected with Angiostrongylus cantonensis. Parasitology. 2021; 148(2): 159–166. PubMed Abstract | Publisher Full Text\n\nChawla S, Jena S, Venkatsan B, et al.: Clinical, pathological, and molecular investigation of Mycoplasma pulmonis-induced murine respiratory mycoplasmosis in a rat (Rattus norvegicus) colony. Vet World. 2017; 10(11): 1378–1382.\n\nWang Y, Liang H, Jin F, et al.: Injured liver-released miRNA-122 elicits acute pulmonary inflammation via activating alveolar macrophage TLR7 signaling pathway. Proc Natl Acad Sci U S A. 2019; 116(13): 6162–6171."
}
|
[
{
"id": "141158",
"date": "22 Jun 2022",
"name": "Yefta Moenadjat",
"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\n1. The abstract\nAbstract reflects the information of the content. The first statement in the background was not in line with the texts elsewhere in the content. In addition, the study was not dealing with the cost and benefit but the efficacy. Then why do the authors put an irrelevant statement here? The method section in the abstract comprises a brief description of the study design and PICO. Consider revising. In the results section of an abstract, it is better to provide the interpretation of the findings in the study rather than providing statistical analysis. In addition, it will be better to put an attribute (for instance, group I, II, II, and control) rather than X1, X2, confusing the readers. Results session: Please consider using English formatting correctly. English does not use a comma but a point for decimals. The abstract's conclusion differs from the conclusion written in the body and is not in line with the study's title and aim, which is focused on the anti-angiogenic effect. Therefore, consider being consistent with it.\n2. Introduction\nThe second paragraph:\nAre the statement in the first three sentences referred to as the same reference as the fourth sentence? There is an interplay of some factors, including VEGF inducing cancer - including HCC - and not solely VEGF. Consider elaborating a little bit regarding this. It does not matter if the authors solely focus on VEGF. Consistency is essential in scientific publication. Therefore, consider using 'level' consistently instead of using a different term of 'concentration' or 'values' in this manuscript.\n\nThe third paragraph:\nIs surgery the most common procedure, or is it a primary or definitive procedure? The author stated: \"...while chemotherapy and targeted therapy are also used.\" The question is: Is it 'also used' or is it the protocol for the HCC?\n\nThe fourth paragraph:\nInstead of providing information from previous studies reporting improvement in the prognosis of HCC, the authors should propose the rationale for using this drug to suppress VEGF. It is essential to answer the question of why use this drug. Authors should provide information on why using the rat model. Particularly the HCC-induced model. There is no information about using DEN in the background that presents the transparency of using DEN to induce HCC. Consider providing such information about toxicity and carcinogenesis effect briefly. Not all readers know about this.\n\nThe fifth paragraph:\nThe statement was not in line with the structure following the checklist. In addition, the link provided indicating the ARRIVE checklist is the same as provided in ARRIVE guidelines https://arriveguidelines.org/ but not specific for this study.\n\n3. Methods\nDescribe systematically and sequentially:\nStudy design, division of groups in randomization (including randomization process, random? who did the randomization? what was the instrument used for this purpose?) Preparation of animals and pre-intervention treatment, Intervention detail in each group, Preparation of materials/materials.\n\nNext, the treatment consists of:\nThe carcinogenesis induction process. Who judges the successful induction, and how did it assess, showing that the intervention was successful? How long does it take to determine that the induction process was successful?\n\nDescribe further detail of tumor-induced DEN characteristics that are similar to an HCC. This section does not explain when Sorafenib and ECGC are given. Either immediately or wait until the HCC induction procedure has been successful. Although, it was explained that the specimens were taken ten weeks after administering these two drugs. The authors did not describe the specific area of the taken specimen but ‘liver tissue’. Was it taken from a tumor mass? Describe further detail of method in systematic order.\n4. Results. Use the same terminology that commented in the introduction and method. Consider providing the findings, but not the interpretation. 5. Discussion. Use the same terminology that commented in the introduction and method. 6. Conclusion. Use the same terminology that commented in the introduction and method.\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": "8814",
"date": "23 Sep 2022",
"name": "Andry Irawan",
"role": "Author Response",
"response": "Dear Dr Yefta Moenadjat, Thank you for allowing us to submit a revised draft of our manuscript titled Anti-angiogenic effect of the combination low-dose sorafenib and EGCG in HCC-induced Wistar rats. We appreciate the time and effort you have dedicated to providing your valuable feedback on our manuscript. We are grateful to the reviewer for their insightful comments on our paper. We have been able to incorporate changes to reflect most of the suggestions provided by the reviewer. We have highlighted the changes within the manuscript. Here is a point-by-point response to the reviewer's comments and concerns. 1. THE ABSTRACT Abstract reflects the information of the content. The first statement in the background was not in line with the texts elsewhere in the content. In addition, the study was not dealing with the cost and benefit but the efficacy. Then why do the authors put an irrelevant statement here? The method section in the abstract comprises a brief description of the study design and PICO. Consider revising. In the results section of an abstract, it is better to provide the interpretation of the findings in the study rather than providing statistical analysis. In addition, it will be better to put an attribute (for instance, group I, II, II, and control) rather than X1, X2, confusing the readers. Results session: Please consider using English formatting correctly. English does not use a comma but a point for decimals. The abstract's conclusion differs from the conclusion written in the body and is not in line with the study's title and aim, which is focused on the anti-angiogenic effect. Therefore, consider being consistent with it. Author’s comment: Thank you for your concern. We have revised the abstract section into a more relevant statement. 2. INTRODUCTION The second paragraph: Are the statement in the first three sentences referred to as the same reference as the fourth sentence? Author’s comment: Yes, they referred to the same reference. There is an interplay of some factors, including VEGF inducing cancer - including HCC - and not solely VEGF. Consider elaborating a little bit regarding this. It does not matter if the authors solely focus on VEGF Consistency is essential in scientific publication. Therefore, consider using 'level' consistently instead of using a different term of 'concentration' or 'values' in this manuscript. Author’s comment: Thank you for your positive feedback. We have elaborated the interplay factors and revised the ‘concentration’ or ‘values’ into ‘level’ terms. The third paragraph: Is surgery the most common procedure, or is it a primary or definitive procedure? Author’s comment: Yes, surgery is the definitive treatment for HCC if it is operable The author stated: \"...while chemotherapy and targeted therapy are also used.\" The question is: Is it 'also used' or is it the protocol for the HCC? Author’s comment: Yes, it is the protocol for unresectable HCC The fourth paragraph: Instead of providing information from previous studies reporting improvement in the prognosis of HCC, the authors should propose the rationale for using this drug to suppress VEGF. It is essential to answer the question of why use this drug. Authors should provide information on why using the rat model. Particularly the HCC-induced model. There is no information about using DEN in the background that presents the transparency of using DEN to induce HCC. Consider providing such information about toxicity and carcinogenesis effect briefly. Not all readers know about this. Author’s comment: Thank you for your considerate question. We have incorporated all the questions stated above within the manuscript. The fifth paragraph: The statement was not in line with the structure following the checklist. In addition, the link provided indicating the ARRIVE checklist is the same as provided in ARRIVE guidelines https://arriveguidelines.org/ but not specific for this study. Author’s comment: The ARRIVE checklist is available in the reporting guideline section. Zenodo: ARRIVE checklist for ‘Anti-angiogenic effect of the combination low-dose sorafenib and EGCG in HCC-induced Wistar rats.’ https://doi.org/10.5281/zenodo.6044890. 3. METHODS Describe systematically and sequentially: Study design, division of groups in randomization (including randomization process, random? who did the randomization? what was the instrument used for this purpose?) Author’s comment: This study was a double-blind, randomized control trial. A laboratory analyst performed randomization into four groups. Preparation of animals and pre-intervention treatment, Intervention detail in each group, Preparation of materials/materials. Author’s comment: Thank you for your insightful comments. We have incorporated the additional statement in the methods section. Next, the treatment consists of: The carcinogenesis induction process. Who judges the successful induction, and how did it assess, showing that the intervention was successful? How long does it take to determine that the induction process was successful? Author’s comment: An anatomical pathologist from Dr Mintoharjo Naval Hospital determined the successful induction under the microscope and required ten weeks to confirm the accomplishment of the induction. Describe further detail of tumor-induced DEN characteristics that are similar to an HCC. Author’s comment: We have elaborated on the similarity of the tumor-induced DEN and HCC characteristics. This section does not explain when Sorafenib and ECGC are given. Either immediately or wait until the HCC induction procedure has been successful. Although, it was explained that the specimens were taken ten weeks after administering these two drugs. Author’s comment: Sorafenib and ECGC were given until the HCC induction procedure was successful by the result from the anatomical pathologist successfully succeeded. The authors did not describe the specific area of the taken specimen but ‘liver tissue’. Was it taken from a tumor mass? Author’s comment: Yes, we took the whole liver tissue-contained tumor masses Describe further detail of method in systematic order. Author’s comment: We have amended the method section more systematically. We look forward to hearing from you in due time regarding the submission, responses, further questions, and comments you may have. Thank you. Sincerely, Andry Irawan"
}
]
},
{
"id": "151185",
"date": "23 Sep 2022",
"name": "Andree Kurniawan",
"expertise": [
"Reviewer Expertise HCC",
"epidemiology",
"cancer",
"hematology"
],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address 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 reported the basic science research regarding the the role of anti angiogenesis in HCC.\nMy comments are:\n1. In the introduction may be added the recent update in HCC since there are emerging new data about anti angiogenesis and immunotherapy . may be added also the role of levantinib. Sorafenib nowadays is not a standard of care for advanced inoperable HCC. Atezo Bev was the standard of care.\n2. Should be added also in the introduction the background rationale for this research since there are several studies about it already.\n3. In the discussion should be added the implication for clinical practice the combination of experimental drugs. What is the implication for translation research from this data?\n4. Advice for further basic research should be added, related to the limitations of this study.\n\n5. In the discussion do not repeat the results, however, compare the results with other studies - similar results or different 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? 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": "8998",
"date": "08 Nov 2022",
"name": "Andry Irawan",
"role": "Author Response",
"response": "Dear Dr Andree Kurniawan, Thank you for allowing us to submit a revised draft of our manuscript titled Anti-angiogenic effect of the combination low-dose sorafenib and EGCG in HCC-induced Wistar rats. We appreciate the time and effort you have dedicated to providing your valuable feedback on our manuscript. We are grateful to the reviewer for their insightful comments on our paper. We have been able to incorporate changes to reflect most of the suggestions provided by the reviewer. We have highlighted the changes within the manuscript. Here is a point-by-point response to the reviewer's comments and concerns. 1. In the introduction may be added the recent update in HCC since there are emerging new data about anti angiogenesis and immunotherapy may be added also the role of levantinib. Sorafenib nowadays is not a standard of care for advanced inoperable HCC. Atezo Bev was the standard of care. Author’s comment: We have incorporated the additional statement in the second last paragraph of the introduction. 2. Should be added also in the introduction the background rationale for this research since there are several studies about it already. Author’s comment: The research rationale has been stated in the 3rd paragraph of the introduction. 3. In the discussion should be added the implication for clinical practice the combination of experimental drugs. What is the implication for translation research from this data? Author’s comment: The combination of low-dose Sorafenib with EGCG has a more potential anti-angiogenic effect in liver cancer by reducing VEGF values compared to the single standard-dose Sorafenib. We can advance the research into human, but firstly we need to examine the toxicity effect in animal research. 4. Advice for further basic research should be added, related to the limitations of this study. Author’s comment: The advice for further studies has been mentioned in the limitation section of the discussion. 5. In the discussion do not repeat the results, however, compare the results with other studies - similar results or different results? Author’s comment: The comparison with other studies has been incorporated in the 4th paragraph of the discussion. We look forward to hearing from you in due time regarding the submission, responses, further questions, and comments you may have. Thank you. Sincerely, Andry Irawan"
},
{
"c_id": "9137",
"date": "19 Dec 2022",
"name": "Andry Irawan",
"role": "Author Response",
"response": "Dear Dr. Yefta Moenadjat, Thank you for allowing us to submit a revised draft of our manuscript. We have adjusted several texts and sentences based on your highlighted texts. We also attempted to register the experimental study; however, all animal study registries must be submitted before the research is done. We sincerely regret this matter, and our evaluation will be for the following animal study. We look forward to hearing from you regarding the submission, responses, questions, and comments you may have. Thank you. Sincerely, Andry Irawan"
}
]
}
] | 1
|
https://f1000research.com/articles/11-289
|
https://f1000research.com/articles/11-1355/v1
|
21 Nov 22
|
{
"type": "Research Article",
"title": "Predictive factors for hypothyroidy after hemithyroidectomy",
"authors": [
"Mohamed Amine Chaabouni",
"Moncef Sellami",
"Esma Jameleddine",
"Rania Kharrat",
"Wadii Thabet",
"Malek Mnejja",
"Boutheina Hammami",
"Sirine Ayadi",
"Imen Achour",
"Ilhem Charfeddine",
"Mohamed Amine Chaabouni",
"Esma Jameleddine",
"Rania Kharrat",
"Wadii Thabet",
"Malek Mnejja",
"Boutheina Hammami",
"Sirine Ayadi",
"Imen Achour",
"Ilhem Charfeddine"
],
"abstract": "Background: Hemithyroidectomy is one of the most common procedures performed. It is used to treat patients with benign unilateral nodules. Hemithyroidectomy results in fewer risks of hypothyroidism and the need for thyroid hormone replacement therapy. The present study was designed to identify potential clinicopathologic risk factors associated with the onset of biochemical hypothyroidism. Methods: We conducted a retrospective review of all patients who underwent hemithyroidectomy between 2004 and 2019. Hypothyroidism was defined as a serum thyrotropin level greater than 5 mIU/L. The patients were analyzed for age, sex, preoperative and postoperative thyroid stimulating hormone (TSH), state, side, and volume of the remaining lobe, and histologic diagnosis. Results: Hypothyroidism was diagnosed in 30.8% of 214 patients. This complication appeared in the first year in 83.3% of the cases. A preoperative TSH level greater than 1.32 mIU/l, a remaining volume of the lobe less than 3 ml, and the presence of thyroiditis were associated with a significant increase in the risk of developing hypothyroidism (p<0.01). There were no significant differences in age, sex, state, and side of the remaining lobe. The mean thyroxine dose was 57 ± 26 micrograms. Conclusions: The risk of hypothyroidism after hemithyroidectomy should be assessed prior to surgery. Close monitoring is recommended in patients at high risk of developing this complication. However, all patients who undergo hemithyroidectomy should be monitored at least for the first year.",
"keywords": [
"Hypothyroidism",
"hemithyroidectomy",
"risk factors",
"thyroiditis"
],
"content": "Introduction\n\nThyroid lobectomy is one of the most common procedures performed. It is used to treat patients with benign unilateral nodules and may be considered sufficient in some cases of patients with unifocal subcentimeter papillary carcinoma without a significant history of risk factors.1 Thyroid lobectomy also helps prevent the risk of hypocalcemia and bilateral recurrent laryngeal nerve paralysis.1,2 Furthermore, it was recognized that, by maintaining functioning thyroid tissue, lobectomy results in fewer risks of hypothyroidism and the need for thyroid hormone replacement therapy.2 For these patients, it was common to start prophylactic suppressive hormonal therapy to prevent the occurrence of a nodular recurrence in the remaining lobe. However, the efficiency of this treatment has come under question, especially since it has significant side effects such as atrial fibrillation and osteoporosis, especially in menopausal women.3 Since leaving this therapy, many cases of hypothyroidism have developed after thyroid lobectomy.\n\nHypothyroidism is an underrated but important complication of thyroid lobectomy with life-threatening side effects. Furthermore, a small percentage of patients on hormone replacement therapy have also been found to continue to complain of fatigue, lack of energy, discrete cognitive disorders, and mood disturbances, despite biochemical euthyroidism.1\n\nTherefore, it is important to determine the risk factors for developing this complication in patients who undergo lobectomy. It will be useful to surgeons, as they will be able to inform patients about this risk, the need for close monitoring, and its duration.2,4 For example, earlier initiation of thyroid hormone replacement therapy may be recommended for high-risk patients.4 Furthermore, a patient with a small nodule (<1cm) in the remaining gland that will be monitored but with a high risk of developing hypothyroidism after lobectomy, the optimal approach would be total thyroidectomy.4\n\nThe purpose of our study was to evaluate the incidence of hypothyroidism after hemithyroidectomy in our patients and to identify risk factors for the development of this complication.\n\n\nMethods\n\nWe conducted a retrospective study of patients who underwent hemithyroidectomy in our department over 15 years (from 2004 to 2019). During this period, preoperative and postoperative TSH were systematically performed.\n\nHemithyroidectomy was defined as unilateral thyroid lobectomy with or without ismethectomy with preservation of the contralateral lobe.\n\nInclusion criteria were normal thyroid function during the preoperative period (normal level of TSH), no preoperative thyrotoxin treatment or any thyroid medications were taken, no systematically administered thyroid hormone replacement after the operation, and at least one postoperative measurement of TSH was required.\n\nPatients in whom we discovered thyroid cancer on the definitive histological examination were also excluded from the study.\n\nPostoperative hypothyroidism was defined by a TSH level > 5 mUI/L at any time during the postoperative period. The normal range of TSH in our Hospital laboratory is 0.25–5 mIU/L.\n\nWe calculated the remaining volume of the gland considering that the lobe has a roughly ellipsoidal shape. A mathematical formula was adopted: Volume = π/6 × (a × b × c) with a, b, and c representing, respectively, the length, width, and depth determined by ultrasound.\n\nWe studied age, sex, preoperative and postoperative TSH levels, operated side, definitive histopathological examination, state of the remaining lobe, follow-up time and the average maintenance dose of Thyroxin for patients who needed thyroid hormone replacement anytime during monitoring.\n\nWe used the receiver operating characteristic (ROC) curve to identify the threshold values of the preoperative TSH level and the volume of the remaining lobe that have the best sensitivity-to-sensitivity ratio to predict postoperative hypothyroidism.\n\nStatistical analysis was done using SPSS 20. We performed the Chi-square test for qualitative variables and the Student t test for quantitative variables. The Mann-Whitney test was used for nonparametric variables.\n\nTo calculate the risk of occurrence of hypothyroidism over time, we use the Kaplan-Meier method.\n\nA p-value < 0.05 was considered statistically significant.\n\nThis study received ethics approval from the Research Ethics Committee of the University Hospital (dated 24 October 2022, approval number 06/2022). In our institution, a retrospective study does not require ethics committee approval. We submitted the study to the ethics committee because it was a requirement of the journal. Written informed consent for publication of their clinical details was obtained from the patients. Written consent to anonymously use patient data for scientific purposes is given by all patients admitted to our department.\n\n\nResults\n\nA total of 232 patients were included.21 Eighteen patients were withdrawn from the study as thyroid cancer was discovered on the definitive histological examination. Of 214 patients studied, 66 (30.8%) developed postoperative hypothyroidism.\n\nThe mean age in the group of postoperative euthyroid patients and the group of postoperative hypothyroidism was, respectively, 44±13 years and 43±17 years (p=0.92).\n\nThe male-to-female sex ratio of the postoperative euthyroid group and the hypothyroidism group was, respectively, 0.09 and 0.06 (p=0.49).\n\nThe mean preoperative TSH level for the euthyroid group and the hypothyroidism group was, respectively, 1.2±0.7 and 2.2±1.3 mUI/L (p<0.001) (Figure 1).\n\nThe threshold value of the preoperative TSH level that has the best sensibility-to-sensitivity ratio was 1.32 mUI/L with a sensitivity of 82% and a sensibility of 62%. The risk of developing postoperative hypothyroidism was statistically higher in the group of patients with a TSH below the limit (p<0.001).\n\nUltrasonography of the thyroid identified seven cases of the remaining hypertrophic lobe, 37 cases of the remaining nodular lobe, and 170 cases of the remaining lobes free from any anomalies. The state of the remaining lobe had no impact on the occurrence of postoperative hypothyroidism.\n\nThe mean volume of the remaining lobe was 4.36±2.3 ml for patients who did not develop postoperative hypothyroidism compared to 3.4±2 ml for those who developed it during follow-up. This difference was statistically significant (p=0.01). According to the ROC curve, we derived a threshold volume for the remaining lobe of 3 mL. This volume has the best sensibility to sensitivity ratio, which were respectively 61.8% and 55%.\n\nIn total, 91 patients had a right lobectomy and 123 patients had a left one. However, the operated side was not a predictive factor for the occurrence of postoperative hypothyroidism.\n\nThe final histopathological examination of the resected sample showed 51 cases of thyroid adenoma and 159 cases of toxic multinodular goiter. The association with lymphocytic thyroiditis was described in 32 cases. Among these histopathological factors, only the presence of thyroiditis was correlated with the risk of developing hypothyroidism (p=0.001). Patients with associated lymphocytic thyroiditis were three times more likely to develop postoperative hypothyroidism (odd ratio: 3.58).\n\nRegarding the time to onset of postoperative hypothyroidism, 62.1% of the patients were diagnosed using laboratory tests in the first 6 months, and 83.3% of the patients within the first year of monitoring (Figures 2 and 3). The average time to the occurrence of this complication was estimated at 10 months (IC 95% 8.5-12.04).\n\nThe mean follow-up duration for the patients was 23 months (4 months – 7 years). Patients who lost follow-up after the first postoperative check-up represented 20.5% of the cases. For patients who had postoperative hypothyroidism, the mean hormonal substitutive dose was 57±26 μg per day.\n\n\nDiscussion\n\nThe half-life of thyroxine produced by the thyroid gland is approximately 7 days. It is recommended to wait at least four to five half-lives of TSH before measuring the TSH level to obtain a precise evaluation of the thyroid hormone produced by the residual thyroid lobe.4 During postoperative monitoring, some patients continue to have normal thyroid function, while in others we noticed an increase in TSH levels. Hypothyroidism can be subclinical with mild TSH levels (TSH from 4.5 to 10 mUI/L) and show a spontaneous return to the euthyroid state due to a compensatory response of the hypothalamic-pituitary-thyroid axis. For patients with a TSH level of less than 10 mUI/L, routine thyroid hormone replacement is not recommended.5 Transient hypothyroidism was found in 33.7 to 67% of the patients. The recovery duration ranges according to the literature from 12 to 18 months.6\n\nHypothyroidism is one of the most important complications of hemithyroidectomy. Its incidence varies between 6 and 50 % depending on the study.2,7 This wide range of incidence can be partially attributed to the different normal reference levels of TSH.1 In a meta-analysis that included 32 studies (4899 patients), Verloop et al. concluded that the risk of developing postoperative hypothyroidy was approximately 22%. A large proportion of those had subclinical hypothyroidism and one in 25 patients developed clinical hypothyroidism.7 In our study, 30.8% of the patients developed biological postoperative hypothyroidy. However, this complication can be overestimated or underestimated as our mean follow-up duration was 23 months and 20.5% of our patients lost follow-up after their first check-up.\n\nThe risk factors attributed to the causation of hypothyroidism in various studies include advanced age, an elevated preoperative TSH level or a normal or high normal value, preoperative hyperthyroidism, autoimmune thyroiditis, multinodular goiter and remnant thyroid volume inferior to 3.2 ml.2,4,8\n\nIn our study, a preoperative TSH level greater than 1.32 mUI/L was a predictive factor for the development of postoperative hypothyroidism with a sensitivity of 82% and a sensibility of 62%. In a meta-analysis performed in 2013, the authors found that patients with a preoperative TSH level greater than 2.5 μUI/L were three times more likely to develop postoperative hypothyroidism.1 The limit for TSH levels varied from one study to another; 2.6 for Ahn et al.,5 2 for Chong et al.,6 and 1.7 for Park et al.9 Said et al. found that for each increase in TSH level of 1 μIU/ml, there is an approximate doubling of the risk of hypothyroidism.8 A higher TSH preoperatively in patients undergoing a hemithyroidectomy indicates a lower reserve of thyroid function and tends to develop hypothyroidism.2 Furthermore, for patients with mild hypothyroidism (TSH level <10), the finding of a TSH level per operative at least 2.6 mIU/L was correlated with a higher risk of developing unrecovered subclinical hypothyroidism.5 Park et al. found that preoperative serum TSH level >1.7 mIU/L was an independent risk factor for postoperative hypothyroidism and persistent hypothyroidism without recovery. They also found that a 1-year postoperative serum TSH level of at least 3.1 mIU/L was an independent factor in predicting late hypothyroidism during follow-up.9\n\nFurthermore, we have observed that lymphocytic thyroiditis within the resected lobe was associated with a statistically higher risk of developing postoperative hypothyroidism.10 Hashimoto thyroiditis is the most frequent autoimmune disease of the thyroid gland.2 Ahn et al. also found in their study a correlation between Hashimoto thyroiditis and the risk of developing postoperative hypothyroidism.11\n\nLymphocytic infiltration of the thyroid gland can lead to a decrease in its function, and a semiquantitative analysis found that as the degree of lymphocytic infiltration within the resected thyroid gland increased, the possibility of postoperative hypothyroidism also increased.12 Furthermore, according to a review of histological data, postoperative TSH levels were found to be significantly associated with lymphocytic infiltration (LI) and germinal center (GC) formation (considered a histological measure of immunologic activation). Specifically, patients with a high LI/GC score had significantly higher mean TSH levels than those with a low LI/GC score.13\n\nThe disadvantage of these scores is their unavailability before the surgery. The level of thyroid antibodies can overcome this problem. Lee et al found that the presence of microsomal antibodies was a significant preoperative predictor for levothyroxine supplementation.14 Other studies found a strong association between detectable levels of thyroid antibodies and histological lymphocyte infiltration in the surrounding thyroid gland.11 The measurement of preoperative anti-thyroperoxidase antibodies can be used as a simple tool to estimate the risk of hypothyroidism in more detail before planning surgery.7 However, in our department, the measurement of thyroid antibodies for simple nodules and unilateral goiter in euthyroid patients (both clinical and biological) was not systematic. Therefore, this predictive factor could not be assessed.\n\nCho et al. found that preoperative TSH between 2.0 and 5.9 mIU/L, and two or more positive factors of Thyroglobulin, anti-thyroglobulin, and anti-thyroid peroxidase (anti-TPO) strongly increase the risk of developing postoperative hypothyroidism.3\n\nThe residual thyroid volume was evoked as a possible predictive factor for postoperative hypothyroidism. Lang et al. found in their prospective study that if the adjusted volume of the body surface area was less than 3.2 ml, the patient is three times more likely to develop this complication. In this same study, the adjusted residual volume of the surface area of the non-body was also a significant risk factor.15 In our study, a residual volume of fewer than 3 ml was correlated with a higher risk of hypothyroidism. Some authors studied the effect of isthmus-preserved thyroid lobectomy and found that the incidence rate of hypothyroidism after this surgery was lower.16\n\nTo predict this complication, some authors established a risk-scoring system. One of these scores was established based on preoperative TSH level and age. The incidence of hypothyroidism was 3% with a risk score of 0, 20% with a score 1, 39% with a score 2, and 70% with a score 3.17 This risk scoring system has the advantage of predicting risk before surgery, allowing the surgeon to discuss it with patients.\n\nThe time to develop clinical hypothyroidism in our study was 12 months for 83.3% of the patients. This is consistent with most of the studies found in the literature, and most patients develop it at 12 months.2 In a study by Al-Shalhoub et al., hypothyroidism was diagnosed in the first 6 months in 70% of patients.18 It appeared in the first 3 months after surgery in 84.5% of cases and after 9 months in 91.2% of patients.5\n\nIdentifying risk factors of the need for thyroid hormone supplementation can help surgeons to provide preoperative counseling to patients and to develop a monitoring plan.19\n\nRegarding postoperative monitoring, there is no algorithm or wide consensus on it. However, it is recommended to obtain a first postoperative TSH measurement for all patients who have undergone hemithyroidectomy 3 months after surgery so compensation by the remaining thyroid lobe may occur.13 Some authors suggest that postoperative TSH should be determined at 6 weeks, 6 months, and 12 months postoperatively.2 For others, thyroid function should be evaluated at 3, 6 months, and 1 year after surgery, then once or twice a year thereafter for low-risk patients. Additional monitoring of thyroid function at 2 and 9 months was optional for high-risk patients.3 Seiberling et al. believe that it may be wise to follow patients with risk factors (including monitoring thyroid function) more closely during the postoperative period.20 For Park et al., patients with high postoperative 1-year TSH levels (3.1 mIU/L) could require more frequent follow-up for thyroid function evaluation, even if they could be euthyroid 1 year after lobectomy.9\n\n\nConclusion\n\nPatients who had a hemithyroidectomy should be closely monitored during the first years after surgery with biological and clinical check-ups one or twice a year. The first follow-up should be 6 months after surgery. The frequency of thyroid function tests and examinations, as well as the proposed timeof the monitoring plan, should be determined depending on the presence or absence of risk factors for developing post-hemithyroidectomy hypothyroidism.",
"appendix": "Data availability\n\nFigshare: Predictive factors for hypothyroidy after hemithyroidectomy, https://doi.org/10.6084/m9.figshare.21404862.v1. 21\n\nThis project contains the following underlying data:\n\n- data.sav (SPSS format; all datasets have been de-identified in accordance with the Safe Harbor method.)\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).\n\n\nReferences\n\nKandil E, Krishnan B, Noureldine SI, et al.: Hemithyroidectomy: a meta-analysis of postoperative need for hormone replacement and complications. ORL J. Otorhinolaryngol Relat. Spec. 2013; 75(1): 6–17. Publisher Full Text\n\nNg P, Ho C, Tan WB, et al.: Predictors of thyroxine replacement following hemithyroidectomy in a south east Asian cohort. Head Neck. 2019; 41(5): 1463–1467. PubMed Abstract | Publisher Full Text\n\nCho JS, Shin SH, Song YJ, et al.: Is it possible to predict hypothyroidism after thyroid lobectomy through thyrotropin, thyroglobulin, anti-thyroglobulin, and anti-microsomal antibody? J. Korean Surg. Soc. 2011; 81(6): 380–386. PubMed Abstract | Publisher Full Text\n\nBeisa V, Kazanavicius D, Skrebunas A, et al.: Prospective Analysis of Risk for Hypothyroidism after Hemithyroidectomy. Int. J. Endocrinol. 2015; 2015: 313971.\n\nAhn D, Sohn JH, Jeon JH: Hypothyroidism Following Hemithyroidectomy: Incidence, Risk Factors, and Clinical Characteristics. J. Clin. Endocrinol. Metab. 2016; 101(4): 1429–1436. PubMed Abstract | Publisher Full Text\n\nChong SS, Hoh SY, Huang SM, et al.: Post-hemithyroidectomy hypothyroidism in non autoimmune thyroiditis patients: Incidence, risk factors and duration of follow up. Asian J. Surg. 2019; 42(11): 957–962. PubMed Abstract | Publisher Full Text\n\nVerloop H, Louwerens M, Schoones JW, et al.: Risk of Hypothyroidism following Hemithyroidectomy: Systematic Review and Meta-Analysis of Prognostic studies. J. Clin. Endocrinol. Metab. 2012; 97(7): 2243–2255. Publisher Full Text\n\nSaid M, Chiu V, Haigh PI: Hypothyroidism after hemithyroidectomy. World J. Surg. 2013; 37(12): 2839–2844.\n\nPark S, Jeon MJ, Song E, et al.: Clinical Features of Early and Late Postoperative Hypothyroidism After Lobectomy. J. Clin. Endocrinol. Metab. 2017; 102(4): 1317–1324. PubMed Abstract | Publisher Full Text\n\nChu KK, Lang BH: Clinicopathologic predictors for early and late biochemical hypothyroidism after hemithyroidectomy. Am. J. Surg. 2012; 203(4): 461–466. PubMed Abstract | Publisher Full Text\n\nAhn D, Lee GJ, Sohn JH: Levothyroxine Supplementation Following Hemithyroidectomy: Incidence, Risk Factors, and Characteristics. Ann. Surg. Oncol. 2019; 26(13): 4405–4413. PubMed Abstract | Publisher Full Text\n\nKoh YW, Lee SW, Choi EC, et al.: Prediction of hypothyroidism after hemithyroidectomy: a biochemical and pathological analysis. Eur. Arch. Otorhinolaryngol. 2008; 265(4): 453–457. PubMed Abstract | Publisher Full Text\n\nJohner A, Griffith OL, Walker B, et al.: Detection and management of hypothyroidism following thyroid lobectomy: evaluation of a clinical algorithm. Ann. Surg. Oncol. 2011; 18(9): 2548–2554. PubMed Abstract | Publisher Full Text\n\nLee DY, Seok J, Jeong WJ, et al.: Prediction of thyroid hormone supplementation after thyroid lobectomy. J. Surg. Res. 2015 Jan; 193(1): 273–278. PubMed Abstract | Publisher Full Text\n\nLang BH, Wong CKH, Wong KP, et al.: Effect of Thyroid Remnant Volume on the Risk of Hypothyroidism After Hemithyroidectomy: A Prospective Study. Ann. Surg. Oncol. 2017; 24(6): 1525–1532. PubMed Abstract | Publisher Full Text\n\nSalih AM: Prevalence of hypothyroidism among patients with isthmus-preserved thyroid lobectomy. J. Int. Med. Res. 2018; 46(9): 3819–3823. PubMed Abstract | Publisher Full Text\n\nTomoda C, Ito Y, Kobayashi K, et al.: Subclinical hypothyroidism following hemithyroidectomy: a simple risk-scoring system using age and preoperative thyrotropin level. ORL J. Otorhinolaryngol. Relat. Spec. 2011; 73(2): 68–71. PubMed Abstract | Publisher Full Text\n\nAl-Shalhoub AK, Al-Dhahri S: Risk Factors of Post-Hemithyroidectomy Hypothyroidism. Saudi J. Med. Med. Sci. 2017; 5(1): 45–48. PubMed Abstract | Publisher Full Text\n\nBuehler LA, Madhun NZ, Bena J, et al.: Hormonal Outcomes Following Hemithyroidectomy. Otolaryngol. Head Neck Surg. 2021; 164(5): 1011–1018. Publisher Full Text\n\nSeiberling KA, Dutra JC, Bajaramovic S: Hypothyroidism following hemithyroidectomy for benign nontoxic thyroid disease. Ear Nose Throat J. 2007; 86(5): 295–299. PubMed Abstract | Publisher Full Text\n\nSellami M, Chaabouni MA:Predictive factors for hypothyroidy after hemithyroidectomy. figshare. [Dataset].2022. Publisher Full Text"
}
|
[
{
"id": "156287",
"date": "28 Nov 2022",
"name": "Ingrid Breuskin",
"expertise": [
"Reviewer Expertise head and neck cancer"
],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 about the predictive factors for hypothyroïdism after loboisthmectomy.\n\nIt shows that preoperative TSH, thyroïditis and the volume of the remaining lobe are predictive.\n\nIt is an interresting work, nicely written and easy to understand.\n\nIt is maybe a bit too descriptive. A table with the different characteristics would be interresting and more visual (age, sex, histological results.)\n\nIt would have also been interesting to analyse what happens after 1 year. As 20% of the patients with hypothyroidism will develop hypothyroidism after this period.\n\nWhy have they excluded patients with cancer? It is because they have all undergone a completion thyroidectomy? If yes ok but if no, they should be integrated.\n\nIn the introduction part, I would not discuss about systematic hormone supplementation for hemithyroidectomy as it is not done anymore, or I would greatly temper the statement.\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": "9119",
"date": "05 Jan 2023",
"name": "Moncef Sellami",
"role": "Author Response",
"response": "We want to thank the reviewer for the quality and relevance of the feedback. This is a study about the predictive factors for hypothyroïdism after loboisthmectomy. It shows that preoperative TSH, thyroïditis, and the volume of the remaining lobe are predictive. It is an interresting work, nicely written and easy to understand. It is maybe a bit too descriptive. A table with the different characteristics would be interresting and more visual (age, sex, histological results.) Author response: A table was added in the results section (Table 1). It would have also been interesting to analyse what happens after 1 year. As 20% of the patients with hypothyroidism will develop hypothyroidism after this period. Author response: Patients with postoperative hypothyroidism who were followed for more than 1 year continued thyroid hormone replacement therapy. Why have they excluded patients with cancer? It is because they have all undergone a completion thyroidectomy? If yes ok but if no, they should be integrated. Author response: Before 2015, the majority of patients with cancer in our department had a total thyroidectomy. Furthermore, after a lobectomy for thyroid cancer, we maintain a TSH target between 0.5 and 2 mIU/L (according to ATA 2015). In the introduction part, I would not discuss about systematic hormone supplementation for hemithyroidectomy as it is not done anymore, or I would greatly temper the statement. Author response: The modification was made according to the recommendation of the reviewer."
}
]
},
{
"id": "156290",
"date": "07 Dec 2022",
"name": "Senda Turki",
"expertise": [
"Reviewer Expertise Rhinology",
"Otology",
"Balance diseases and vertigo",
"Sleep Apnea",
"Neck surgery"
],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis article addresses a particularly important topic. Indeed, the risk factors for hypothyroidism after hemithyroidectomy must be evaluated precisely, given the fact that it’s a frequent surgical option for managing several unilateral thyroid diseases. There are several articles about this topic in the literature and different factors have been identified.\nThis study is interesting, easy to follow and clear to understand. The series studied is important with a well conducted statistical study.\nHowever, I have a few comments that the authors might consider:\n\n1. The introduction is too long (for example: the part about systematic hormone supplementation should be displaced to the beginning of the discussion).\n\n2. Methods:\n“Benign definitive histological examination” should be added to the inclusion criteria. Indeed, patients with papillary cancer aren’t initially included in the study. They aren’t excluded from it. Moreover, why were these patients not included (several low-risk papillary cancers can be treated by hemithyroidectomy)?\n\nThe ROC’s explanation is unclear, especially “the sensitivity-to-sensitivity ratio” which is also mentioned in the results part: “The Receiver operating characteristic (ROC) curves compare sensitivity versus specificity across a range of values for the ability to predict a dichotomous outcome.” (Statistic courses).\n\n3. Results:\nThe risk of developing postoperative hypothyroidism was higher in the group of patients with a TSH above the limit, not below.\n\nThere is no information regarding your patients’ clinical symptoms of hypothyroidism.\n\nYou could have added figures and tables for the description of your series.\n\n4. Discussion:\n\"In our study, a preoperative TSH level greater than 1.32 mUI/L was a predictive factor for the development of postoperative hypothyroidism with a sensitivity of 82% and a sensibility of 62%.”: do you mean 'specificity' instead of 'sensibility'?\n\nIn the literature, the female sex is also a risk factor.\n\n5. You could have added in the reference a recent meta-analysis: Li et al. (20201).\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": "9120",
"date": "05 Jan 2023",
"name": "Moncef Sellami",
"role": "Author Response",
"response": "We thank the reviewer for the constructive comments that demonstrated interest in the topic of our manuscript. 1. The introduction is too long (for example: the part about systematic hormone supplementation should be displaced to the beginning of the discussion). Author response: As recommended by the reviewer, we have shortened the Introduction section. 2. Methods: “Benign definitive histological examination” should be added to the inclusion criteria. Indeed, patients with papillary cancer aren’t initially included in the study. They aren’t excluded from it. Moreover, why were these patients not included (several low-risk papillary cancers can be treated by hemithyroidectomy)? Author response: We initially included all patients who underwent lobectomy. Patients in whom we discovered thyroid cancer on the definitive histological examination were excluded from the study. In the results section, a total of 232 patients were included and 18 patients were excluded from the study because thyroid cancer was found at the definitive histological examination. Based on the recommendations (ATA 2015) we maintain an initial TSH goal between 0.5 and 2 mU/L after lobectomy for low-risk cancer and we cannot identify postoperative hypothyroidism. The ROC’s explanation is unclear, especially “the sensitivity-to-sensitivity ratio” which is also mentioned in the results part: “The Receiver operating characteristic (ROC) curves compare sensitivity versus specificity across a range of values for the ability to predict a dichotomous outcome.” (Statistic courses). Author response: There was a typographical error. Indeed, receiver operating characteristic (ROC) curves compare sensitivity versus specificity over a range of values. 3. Results: The risk of developing postoperative hypothyroidism was higher in the group of patients with a TSH above the limit, not below. Author response: Correction was made. There is no information regarding your patients’ clinical symptoms of hypothyroidism. Author response: These patients did not manifest clinical signs of hypothyroidism. These data were included in the results section. You could have added figures and tables for the description of your series. Author response: We included a table describing patient characteristics (table 1). 4. Discussion: \"In our study, a preoperative TSH level greater than 1.32 mUI/L was a predictive factor for the development of postoperative hypothyroidism with a sensitivity of 82% and a sensibility of 62%.”: do you mean 'specificity' instead of 'sensibility'? Author response: Correction was made. Effectively, it is the’specificity’. In the literature, the female sex is also a risk factor. Author response: We have added this information with the reference suggested by the reviewer. 5. You could have added in the reference a recent meta-analysis: Li et al. (20201). Author response: The reference suggested by the reviewer was included."
}
]
}
] | 1
|
https://f1000research.com/articles/11-1355
|
https://f1000research.com/articles/11-291/v1
|
09 Mar 22
|
{
"type": "Study Protocol",
"title": "Effectiveness of Gatekeeper Training Program (GTP) on awareness, attitude, mental help seeking intention and gatekeeper behavior among Koraga tribe: A study protocol",
"authors": [
"Flavia Sharlet Noronha",
"Tessy Treesa Jose",
"Anice George",
"Linu Sara George",
"Flavia Sharlet Noronha",
"Anice George",
"Linu Sara George"
],
"abstract": "Aim: This study aims to build the capacity of the people at grass root level as gatekeepers of mental health. It will assess the effectiveness of the Gatekeeper Training Program (GTP) on gatekeeper behaviour, awareness, attitude, and mental help seeking intention. Design: An evaluative research approach in two phases. Phase 1: Cross-sectional house-to-house exploratory survey. Phase 2: A quasi-experimental design with multiple follow ups at 0, 6 and 12 months. Method: Data will be collected using standardized tools like Mental Health Knowledge Questionnaire (MHKQ), Community Attitude towards Mentally Ill (CAMIS), Mental Help Seeking Intention (MHSIS) and Gatekeeper Behavior Scale (GBS). For Phase 1, a house-to-house survey will be conducted among the selected colonies of Koraga tribe to determine their awareness, attitude, and mental help seeking intention regarding common mental health problems. Phase 2 includes identification of the leaders/representatives of the selected tribal colonies, and involving them in GTP. Pre-test and multiple post-test will be conducted in Phase 2 at 0, 6, 12 months. The study is funded by Indian Council of Medical Research from 16 August 2021 for 3 years duration. Discussion: Treatment gap in psychiatric disorders remains an issue of great concern. Evidence based research promotes task shifting approaches in dealing with mental health problems in the community. Capacity building programs like GTP for the underprivileged section of the society are important especially in low and middle income group of countries. Impact: This need based GTP, will ensure mental health first aid in the society. Early identification of people with mental health problems at their doorsteps has huge impact on the prognosis of the illness, closing the treatment gap and stigma reduction.",
"keywords": [
"Koraga",
"Tribal",
"Gatekeeper behaviour",
"mental help",
"attitude",
"mental health problems",
"mental health knowledge",
"GTP"
],
"content": "Introduction\n\nTribal communities in India cannot be classified as one homogenous group, as they vary at different levels of development and belong to other ethnic-lingual groups. There are around 42,48,987 tribal people in Karnataka, of whom 50,870 belong to the primitive group. This primitive group of the tribal population represents 6.95 percent of the total population of Karnataka. The Koraga tribe is one of the most backward tribes classified under Particularly Vulnerable Tribal Group (PVTG) as declared by the Government of India. This tribe is scattered over many districts of the State, particularly in Udupi and Dakshina Kannada. Their number is 14,794 as per the 2011 census. The occupation of the Koraga tribe is at the pre-agricultural stage of development, i.e., they are experts in basket weaving, which is the primary source of income, but presently majority of them work as daily wage laborers (Pujar et al., 2017). Poverty and illiteracy are the main problems in their community (Roy et al., 2015). They are the poorest among the scheduled tribes of Karnataka (Nalinam, 2013); around 48.4% of Koraga population have a meager monthly income of Rs.2001-4000 (Pujar et al., 2017). Alcoholism is another threat to the mental well-being of the Koraga tribal members. Several studies have reported widespread alcoholism among Koraga men and women; they also indulge in smoking beedi (tobacco) and chewing betel leaves. They spend a significant portion of the paltry income, leaving very little for other day-to-day expenses (Nalinam, 2013). Even after 72 years of Independence and 60 years of the abolition of the untouchability practice, primary health care delivery is still inaccessible to the most vulnerable, primitive population. Unfortunately, people from Koraga tribe are still the “untouchables among the untouchable” (Balakrishnan & Sharma, 2012).\n\n\nBackground\n\nIn the Toto tribe of the Sub-Himalayan region, the prevalence rate of mental illness was 48.97%, with depression being the highest (Ghosh et al., 2004). A recent study from the West Godavari district of Andhra Pradesh, predominantly comprising people from scheduled tribes, highlighted that 15% of the present generation of the tribal population was affected by common mental disorders like stress, depression, suicide risk, and anxiety (Maulik et al., 2017). According to National Health and Family Survey (NHFS) 3, 72% of tribal men aged 15-54 years were addicted to tobacco, and 50% of tribal men were addicted to alcohol (Ministry of Health and Family Welfare & Ministry of Tribal Affairs, 2013). Psychosocial problems were also reported among high school children of the Mysuru region. About 23.7% of children in the tribal area had anxiety disorders, 1.6% had mood disorders, 3.2% had suicidality, and 2.2% were diagnosed with ADHD (Pradeep et al., 2018). Koraga, a particularly vulnerable tribal group of Udupi district has shown significant liver dysfunction due to chronic alcoholism among its population, irrespective of its gender. Most of the daily wages were spent on alcohol as evident by majority (75%) of them being alcoholics (Mungli et al., 2013). Several studies have found that in general there is a vast treatment gap when it comes to the accessibility of mental health services. Although there are several reasons for the same, lack of awareness and stigma towards mental illness and mentally ill people are the most prominent. A cross-sectional survey assessing the effectiveness of community mental health programs in a tribal area of South India revealed that the mean score on awareness regarding mental illness was 5.13±2.27 among its study population (Yalsangi, 2011). However, contrary to this, the majority (64%) of people from Naga tribe could recognize the presence of a mental health problem in a depicted vignette (Longkumer & Borooah, 2013). Another study on stigma in mental illness revealed that 38.5% of persons with severe mental illness (PSMI) were found poor, and this poverty was strongly associated with stigma related to mental illness (OR 2.60, 95% CI 1.27 to 5.31), and scheduled tribes (2.39, 1.39 to 4.08). Therefore, it was concluded that females or people from lower castes having severe mental illness were more at risk to be economically poor due to stigma related to mental illness (Trani et al., 2015).\n\nThe Systematic Medical Appraisal, Referral and Treatment (SMART) mental health project of Andhra Pradesh found that 7% of people go to faith healers and religious leaders at the very beginning of their mental illness for treatment (Maulik et al., 2017). The preference for magico-religious healing and traditional practices to overcome mental health concerns is prevalent in South-Asian countries, evidenced by a qualitative study from Bangladesh on tribal communities (Rahman et al., 2012). As mentioned earlier, the study poses a challenge to the healthcare delivery system in low-income countries, which mainly caters to the plainland population. A randomized controlled trial across 32 colleges of USA evaluated the effectiveness of the gatekeepers training program and found that trainees' self-perceived knowledge, ability, and confidence to identify students in distress were increased (Lipson et al., 2014). In the long-term effects of community gatekeeper training, 15 (n=40) participants had helped someone at risk of suicide; also, intentions to help and confidence to identify someone at risk of suicide remained high (Deane et al., 2006). Though common mental health problems are prevalent among the tribal population, as evident by the research studies mentioned above, very little is done to improve their condition in India. Hence, it is vital to improve the mental health care system for our most primitive and underprivileged tribal people and develop effective mental health care programs at the grassroots level.\n\nAims and Objectives: The objectives of the study are to:\n\n• Determine the awareness, attitude and mental help seeking intention regarding common mental health problems among the tribal population using standardized tools.\n\n• Assess the awareness, attitude, mental-help seeking intention and gatekeeper behavior among the leaders/representatives of Koraga tribe regarding common mental health problems using standardized tools.\n\n• Find out the effectiveness of gatekeeper training program on awareness, attitude, mental help seeking intention and gatekeeper behavior among the leaders/representatives of Koraga tribe regarding common mental health problems.\n\n• Determine the number of vulnerable individuals identified, counselled, referred to a tertiary center as and when needed by the trained gatekeepers of the tribal community at 12 months of follow up.\n\n\nMethods\n\nThe study is based on an evaluative approach to meet its objectives. The study will be done in two phases. Phase 1 will be a cross-sectional house-to-house survey where data will be collected from the Koraga tribe and Phase 2 will have a quasi-experimental design which includes an experimental and a control group. There will be pre-test and post-test for both the groups with multiple follow ups at 0, 6 and 12 months. GTP (intervention) will be carried out only for the experimental group.\n\nThe study will be conducted in Koraga tribal colonies of Udupi district, Karnataka. There are around 360 colonies spread across seven blocks of the Udupi district, and each colony comprises a minimum of 8-10 families. The colonies are geographically distinct in nature. The total Koraga population is 11,133 in Udupi district. Variables under Phase 1 of the study will be awareness, attitude, and mental help seeking intention and Phase 2 variables include awareness, attitude, mental help seeking intention and gatekeeper behavior regarding common mental health problems.\n\nPhase 1: Based on convenient sampling technique, out of the seven blocks, three blocks will be taken up for the survey. Each of these three blocks comprises a minimum of 35 colonies, hence 25-27 colonies will be selected randomly from each block. Stratified sampling technique (age and gender) will be used to select five samples/colony for the survey.\n\nFor phase 2, convenient sampling technique will be used to select the block (largest block) for experimental and control group. These largest blocks consist of 85-90 colonies, hence 55 colonies will be selected randomly. Further purposive sampling technique will be used to select the participants for GTP (Figure 1: Schematic representation of sampling technique).\n\nPhase 1 sample size is calculated using the formula as mentioned below. In this formula the confidence level is 95%, hence Z2 = 1.96. Here proportion of the tribal people having awareness regarding common mental health problems was assumed to be 0.5 and margin of error (d) = 0.05.\n\nFor Phase 2: Using G* power software the sample size was estimated to be 86 (43 in each group).\n\nWith 20% attrition rate n = 108 (54 in each group).\n\n(Here the values are as follows: Effect size = 0.25, Alpha error = 0.05, Beta error = 0.8, Number of groups = 2, Number of measures = 3.)\n\nPhase 1: People aged between 18-60 years will be selected in a stratified manner (age and gender) and will be included in the study. For phase 2, (both experimental & control groups) block leaders and representatives from each colony who can read & write Kannada and are willing to participate will be included in the study. The exclusion criteria for both Phase 1 & 2 will be those individuals who have attended previous mental health workshops and those who have any acute/chronic illness.\n\nFor Phase 1 data will be collected using standardised tools like Mental Health Knowledge Questionnaire (MHKQ), Community Attitude towards Mentally Ill scale (CAMIS), and Mental Help Seeking Intention scale (MHSIS). Administrative permissions for the data collection is obtained. The data will be collected from the participants after explaining the participant information sheet and obtaining informed consent. Data collection will be carried over six months due to geographical constraints.\n\nFor Phase 2, identification of the participants for the GTP i.e the leaders/representatives of the selected tribal colonies will be done with the help of Department of Tribal Welfare, at the district administrative office. The project staff will approach the selected leaders/representatives of the tribal colonies, explain the participant information sheet, obtain consent and then proceed with data collection. Data for pre-test and multiple post-test (at 0, 6, 12 months) will be collected using standarised tools such as MHKQ, CAMIS, MHSIS and Gatekeeper Behavior Scale (GBS). The project staff will be present during the data collection procedure in both phases of the study.\n\nPermission from the authors of the following standardised tools (except socio-demographic) are obtained.\n\nTool 1: Background information consists of 15 items related to the socio-demographic characteristics of the participants. This tool is developed by the researcher and subjected to validation by experts.\n\nTool 2: The Mental Health Knowledge Questionnaire is a dichotonoums scale consisting of 25 items regarding substance use disorder, depression, suicide, stress, anxiety, its treatment and prevention. Higher the scores attained on this scale indicates greater mental health literacy. This scale was developed by Hanhui Chen, Zhizhong Wang and Michael Roberts in 2013.\n\nTool 3: The Community Attitude towards Mentally Ill scale is 40 item Likert scale measuring the attitude towards mental illness and mentally ill people under four dimensions namely authoritarianism, benevolence, social restrictiveness, and community mental health ideology. Higher the score on CAMIS, indicates greater stigma toward mental illness and a mentally ill person. This scale was developed in 1970’s by Martin Taylor and Michael Dear, Canada.\n\nTool 4: Mental Help Seeking Intention scale (MHSIS) is a 3-item instrument designed to measure respondents’ intention to seek help from a mental health professional if they had a mental health concern with responses ranging from strongly agree (7) to strongly disagree (1). The resulting mean score will range from a minimum of 1 to a maximum of 7. A higher score indicates a greater intention to seek help. This scale is an adapted version from Ajzen’s Theory of Planned Behaviour (2006) and was standardised by Dr Joseph H Hammer and Douglas Spiker in 2018.\n\nTool 5: Gatekeeper Behavior Scale is a Likert scale consisting of 11 items evaluating the preparedness (5 items), likelihood (2 items), and self-efficacy (4 items) of an individual’s gatekeeping skills. Higher scores indicate better gatekeeping skills of the individual. This scale was developed by Albright, G., Davidson, J., Goldman, R., Shockley, K. & Mitchell-Timmons, J. in 2014.\n\nGatekepeer Training Program (GTP) is a two-day mental health workshop conducted at various community locations (as per the convenience of the participants) for the experimental group. The investigator will be in contact with the participants throughout the post-intervention period to review/clarify the issues faced by the gatekeepers, and a diary will be provided to the participants in which they will have to document the details of interactions (if they had) with people having mental health concerns. (The sessions for the two-days workshop is listed in Table 1.) The control group in the study will receive a module on the theoretical concepts of common mental health problems and gatekeeper behaviour. For both the experimental and control group, post-test will be conducted at three points of time i.e., at 0, 6, 12 months. After the follow-up duration of one year, a half-day workshop will be conducted for the control group at various community locations (as per the convenience of the participants). This workshop will focus on the theoretical concepts of the module provided earlier.\n\nValidity and reliability: All the tools (except socio-demographic proforma) that will be used in this study are standardised tools and are validated internationally. Further, to check for cross cultural validity, the tools were validated by a panel comprising of experts from psychiatry, psychiatric social work, community medicine and nurisng. The experts agreed that the tools could be used in a tribal population. The internal consistency of the tools will be measured by appropriate reliability tests by administering the tools to 20 participants from the Koraga community.\n\nEthical consideration: Permission to conduct the study is received from the Head of the Institution, Institution Review Committee and Institutional Ethics Committee (322/2020). Permission is also obtained from the tribal community authority (Integrated Tribal Development Office) at the district government office of Udupi. This study is registered in Clinical Trial Registry of India (CTRI/2021/06/034037 [Registered on: 07/06/2021]. Informed consent will be obtained from all participants, and a complete explanation of the investigation will be provided.\n\nData analysis: Demographic characteristics of the study participants will be presented in descriptive summary tables. The outcome variables across the follow up period will be analyzed by repeated measures of ANOVA. The categorical variables will undergo bivariate analysis by applying the Chi-square test for categorical variables. A p-value of less than 0.05 will be considered the criteria for statistical significance. Overall SPSS 26 version will be used to analyse the data gathered.\n\nDissemination of results: The results will be disseminated via presentations at appropriate scientific conferences and workshops. The findings will also be published in peer-reviewed journals, professional and institutional repositories etc. The result will be discussed with the Department of Tribal Welfare and other stakeholders for improvement of mental health awareness, accessibility to meantal health care and appropriate utilization of community-based resources.\n\n\nDiscussion\n\nThe health status of the tribal population is in a pitiable condition (“Tribal Health,” n.d.). They continue to suffer due to inequity and inaccessibility in the health care system (MOHFW&MOTA, 2013). Traditional healers and magico-religious practices are still popular among the tribal population worldwide (Beals et al., 2005). The stigma associated with mental illness is another cause for the treatment gap in psychiatry. Innovative and decentralized community-based approaches should be adopted to reduce barriers in vulnerable populations. World Health Organization has recognized informal community care by peers or community people as an effective and low-cost method of providing mental health services to the vulnerable population (The Optimal Mix of Services, 2007). Atmiyata, a community-based intervention carried out in Gujarat, India, which provides support to people experiencing mental health issues, found significant improvement in recovery rates and overall quality of life (ATMIYATA: A Community-Led Intervention in Rural India|Mental Health Innovation Network, n.d.). Nimgaonkar and Menon (2015) in their community-based task-shifting program found that the self-referrals rates increased from 27% to 57%. Also, there was positive growth in knowledge, attitude, and practice about mental health. Hence much more research is required in these areas, and best practices in the community needs to be identified. Community-based interventions incorporating the needs, active participation, socio-cultural beliefs, and resources of its people are reportedly successful.\n\nThe desire to commit oneself to social service and help out a person with mental health issues (gatekeeper) vary from person to person and are very subjective in nature. Also in this study the gatekeeper behaviour in the community will be greatly affected by the acceptance of the services despite the stigma, the gatekeepers' belief system, and inter-sectorial coordination among the community stakeholders. Interaction between the gatekeeper and an individual with mental health problems in the community will be collected as a self-report as the researchers cannot reach at the point of time in the community.\n\n\nConclusion\n\nThe information obtained on awareness, attitude, and mental-help-seeking intention regarding common mental health problems will help to build the efficacy of Koraga tribe members in terms of gatekeeping behavior. This study believes that community-based interventions are an integral part of mental health services. Programs that help to build the capacity of the people at the grassroot levels will help in early identification, and referral of individuals with signs and symptoms of possible mental health problems, thus reducing the treatment gap, stigma and making mental health services more accessible.\n\nStudy status:\n\nThe researcher is yet to start with the data collection for the study. At present, the tools were validated for cross cultural validity, and pilot phase of the survey is being planned.\n\nNo underlying data is associated with this article.\n\nExtended data available at Figshare repository: The following datasets are available with their doi.\n\n1. Informed Consent for Phase 1. (Noronha et al., 2022a).\n\n2. Informed Consent for Phase 2. (Noronha et al., 2022b).\n\n3. Participant information sheet for Phase 1. (Noronha et al., 2022c).\n\n4. Participant Informant Sheet - Phase II (Experimental group). (Noronha et al., 2022d).\n\n5. Participant Information Sheet - Phase 2 for Control group. (Noronha et al., 2022e).\n\n6. Background information. (Noronha et al., 2022f).\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\nAuthors contributions\n\n\n\n• Flavia Sharlet Noronha: Conceptualization, Methodology, Funding Acquisition, Validation, Writing – Original Draft Preparation\n\n• Treesa Jose: Conceptualization, Methodology, Funding Acquisition, Project Administration, Supervision, Validation, Visualization, Writing – Review & Editing\n\n• Anice George: Conceptualization, Methodology, Writing – Review & Editing\n\n• Linu Sara George: Conceptualization, Methodology, Project Administration, Writing – Review & Editing",
"appendix": "References\n\nATMIYATA: A community-led intervention in rural India|Mental Health Innovation Network : n.d. Retrieved July 12, 2021. Reference Source\n\nBalakrishnan B, Sharma P: The Koraga Tribe–Untouchables among the untouchables – The Manipal Journal. The Manipal Journal. 2012. Reference Source\n\nDeane FP, Capp K, Jones C, et al.: Two-Year Follow-Up of a Community Gatekeeper Suicide Prevention Program in an Aboriginal Community. Aust. J. Rehabil. Couns. 2006; 12(1): 33–36. Publisher Full Text\n\nGhosh A, Banerjee G, Biswas D: Psychiatric Morbidity in a Sub-Himalayan Tribal Community: An epidemiological Study. Indian J. Psychiatry. 2004; 46(4): 324–332. PubMed Abstract Reference Source\n\nLipson SK, Speer N, Brunwasser S, et al.: Gatekeeper Training and Access to Mental Health Care at Universities and Colleges. J. Adolesc. Health. 2014; 55(5): 612–619. PubMed Abstract | Publisher Full Text\n\nLongkumer I, Borooah IP: Knowledge about and attitudes toward mental disorders among Nagas in North East India. IOSR Journal of Humanities and Social Science (IOSR-JHSS). 2013; 15(4): 41–47. Publisher Full Text Reference Source\n\nMaulik PK, Kallakuri S, Devarapalli S, et al.: Increasing use of mental health services in remote areas using mobile technology: a pre–post evaluation of the SMART Mental Health project in rural India. J. Glob. Health. 2017; 7(1): 010408. PubMed Abstract | Publisher Full Text\n\nMinistry of Health and Family Welfare & Ministry of Tribal Affairs, G. of I: Report of the Expert committee on Tribal Health: Tribal Health in India. 2013. Reference Source\n\nMungli P, Shetty J, Vanishree B, et al.: Cross-sectional study to know the prevalence of liver dysfunction, anemia and metabolic syndrome in the Koraga community in Udupi district of South Karnataka, India. International Journal of A J Institute of Medical Sciences. 2013; 2(1): 3–12. Reference Source\n\nNalinam M: Depopulation of Koraga Tribes in South India. IOSR Journal of Humanities and Social Science (IOSR-JHSS). 2013; 8(4): 1–4. Reference Source\n\nNimgaonkar AU, Menon D: A task shifting mental health program for an impoverished rural Indian community. Asian J. Psychiatr. 2015; 16: 41–47. PubMed Abstract | Publisher Full Text\n\nNoronha FS, et al.: Informed Consent for Phase 1.doc.figshare. Online Resource. 2022a. Publisher Full Text\n\nNoronha FS, et al.: Informed Consent for phase 2.doc.figshare. Online Resource. 2022b. Publisher Full Text\n\nNoronha FS, et al.: Participant information sheet for phase 1.doc.figshare. Online Resource. 2022c. Publisher Full Text\n\nNoronha FS, et al.: Participant Informant Sheet - Phase II (Experimental group). doc. figshare. Online Resource. 2022d. Publisher Full Text\n\nNoronha FS, et al.: Participant Information Sheet - Phase 2 for Control group. doc. figshare. Online Resource. 2022e. Publisher Full Text\n\nNoronha FS, et al.: Background information. doc. figshare. Online Resource. 2022f. Publisher Full Text\n\nPradeep T, Narayana M, Praveen K: Factors Affecting Mental Abnormalities among High School Children in Tribal, Rural and Urban Mysuru, Karnataka. National Journal of Community Medicine. 2018; 9(4): 255–259.\n\nPujar A, Hoogar P, Basavanagouda TT: Socio-Economic Status among Koraga Tribe of Udupi District. Int. J. Interdiscip. Multidiscip. Stud. 2017; 4(2): 147–148. Reference Source\n\nRahman SA, Kielmann T, McPake B, et al.: Healthcare-seeking behaviour among the tribal people of Bangladesh: Can the current health system really meet their needs?. J. Health Popul. Nutr. 2012; 30(3): 353–365. Publisher Full Text Reference Source\n\nRoy S, Hegde HV, Bhattacharya D, et al.: Tribes in Karnataka: Status of health research. Indian J. Med. Res. 2015; 141(5): 673–687. PubMed Abstract | Publisher Full Text | Free Full Text\n\nThe optimal mix of services: Geneva, World Health Organization. Mental Health Planning and Service Development.2007. Reference Source\n\nTrani JF, Bakhshi P, Kuhlberg J, et al.: Mental illness, poverty and stigma in India: A case-control study. BMJ Open. 2015; 5(2): e006355. PubMed Abstract | Publisher Full Text\n\nYalsangi M: 2011. Evaluation of a Community Mental Health programme in a tribal area-South India (master’s thesis) [Achutha Menon Centre for Health Science Studies, Sree Chitra Tirunal Institute for Medical Sciences and Technology, Thiruvananthapuram, Kerala]. Reference Source"
}
|
[
{
"id": "140324",
"date": "20 Sep 2022",
"name": "Dr Mohit Varshney",
"expertise": [
"Reviewer Expertise Mental health",
"Addictive 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\nThis study aims to focus on gatekeeper training and its impact on identifying mental illnesses.\nA pertinent study with rigorous methodology. Can include details of training for application of instruments in local languages in methodology (since most are in English). And how will respondent confidentiality be maintained during the interview? What will happen to those who agree initially to be gatekeepers but than drop out?\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": "8895",
"date": "04 Nov 2022",
"name": "Tessy Treesa Jose",
"role": "Author Response",
"response": "Respected Sir Thank you for reviewing our work and providing imperative suggestions. We shall incorporate your suggestions with apt explanation in our methodology of the study."
},
{
"c_id": "9113",
"date": "09 Jan 2023",
"name": "Tessy Treesa Jose",
"role": "Author Response",
"response": "1. Include details of training for application of instruments in local languages in methodology. The tools used in this study are standardized structured questionnaires available in English language. Since the study population is local tribal population, the tools are translated into the language known by the tribals (Kannada). Re-translation back to English is done by language experts. Since the primary investigator is fluent and well versed in the local language (Kannada), extensive training was not required for the application of tools in local languages. 2. How will respondent confidentiality be maintained during the interview? The consent forms will be coded, and any identification data will be concealed thoroughly for both phase 1 and phase 2. The data collection for phase 1 will be done through house-to-house survey using structured questionnaires and data collection for phase 2 of the study will be done during the GTP, that is, the two-day workshop. Structured questionnaires will be used to collect the data. There is no in-depth interviews or verbatim reports involved in this study, however if the participants need any clarifications while filling out the structured tools, the primary investigator will be there to clarify it. 3. What will happen to those who agree initially to be gatekeepers but then drop out? Participation in this study is voluntary; the participants may decline to participate at any time, and they need not give any reason for the same, and such withdrawal shall be without penalty. If they withdraw from the study before data collection is completed, the data collected until they indicated withdrawal will be used in the study report. The sample size is calculated by keeping in mind the attrition rate at 20%, hence the sample size will not be affected"
}
]
},
{
"id": "154892",
"date": "01 Dec 2022",
"name": "Omar S. H. Al Omari",
"expertise": [
"Reviewer Expertise Mental 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\nDear Authors,\nI have some comments for you to consider before starting your study.\n\n(d) representing the effect size and not the margin of the error; please correct it accordingly. Please revisit the formula and ensure the terms that they used.\n\nSame issue with the second formula. That is, 0.8 represents the power and not the beta error. Please let the statistician help you with this.\n\nI don't know if the researchers used the original English survey or translated version. If so, please comment on the translation process.\n\nI am still trying to understand whether the program's content has been reviewed by a panel of experts or no. If yes, please mention the process. Researchers need to mention the program delivered in one place or different places and to comment on the inter-rater reliability and intra-rater reliability.\n\nThe reference below will help the researchers in planning their sample size and will acquaint them with helpful information about inter-rater and intra-rater reliability.\nWarm Regards,\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? No",
"responses": [
{
"c_id": "9108",
"date": "09 Jan 2023",
"name": "Tessy Treesa Jose",
"role": "Author Response",
"response": "Dear Dr. Omar S. H. Al Omari Thank you for reviewing our work. We will look into the suggestions and work on it and also we appreciate for providing a credible source of reference. Regards."
},
{
"c_id": "9114",
"date": "09 Jan 2023",
"name": "Tessy Treesa Jose",
"role": "Author Response",
"response": "1. Thank you for the suggestion. The formula is modified accordingly in the updated version of the article. 2. The tools used in this study are standardized tools available in English language. Since the study population is local tribal population, the tools are translated into the language known by the tribals (Kannada). Re-translation back to English was done by language experts. Since the primary investigator is fluent and well versed in the local language (Kannada), extensive training was not required for the application of tools in local languages. Both the English and Kannada versions were subjected to cross cultural validity through experts in the field of psychiatry, psychiatric nursing, psychiatry social workers, and clinical psychologist. These experts have extensive experience in working with the people of Koraga tribe. 3. The content for the workshop (module) is being prepared by the primary investigator. Knowledge gap observed from phase 1 (survey) of the study is included in the module. This module will be given to a panel of experts in various fields of psychiatry at local, national, and international level. The suggestions and modifications will be incorporated accordingly. The program will be implemented by the same multi-disciplinary team members in various community locations as per the convenience of the participants or at a single location if all participants (54) could meet up conveniently. Inter-rater and intra-rater reliability is not applicable to this study as only the primary investigator will be recording the data throughout the study period and this study involves only a single trial with follow ups. However, the tools used in this study were tested for reliability and the values are uploaded in the new version of the article."
}
]
}
] | 1
|
https://f1000research.com/articles/11-291
|
https://f1000research.com/articles/11-1549/v1
|
21 Dec 22
|
{
"type": "Data Note",
"title": "AFHIRIS: African Human Iris Dataset (Version 1)",
"authors": [
"Oluwatobi Akande",
"Nzube Ojimba",
"Atele Oghenekaro",
"Oluwakemi Abikoye",
"Roseline Ogundokun",
"Akinyinka Akindele",
"Nzube Ojimba",
"Atele Oghenekaro",
"Oluwakemi Abikoye",
"Akinyinka Akindele"
],
"abstract": "Biometric systems remain the most widely used methods for identification and authentication purposes. Their wide acceptability has opened up more research into new application areas of biometric systems. However, biometric research requires an appropriate biometric dataset to validate the proposed technique. This dataset could be privately owned or publicly available for research purposes. In the field of iris biometric research, the iris dataset produced by the Chinese Academy of Sciences (CASIA) is the first, most popular, and widely used publicly available iris dataset. However, the increasing popularity and acceptability of human iris-related research have called for additional benchmarks, and therefore, new publicly available databases of human iris images. Existing publicly available human iris datasets have been collected from non-African subjects; therefore, this dataset is the first publicly available human iris dataset of African descent. Three categories of images were collected from 1028 volunteers that participated in the data collection task. The first category was made up of four iris images that were captured when the volunteers used spectacles, while the second category includes four iris images that were captured when the volunteers wore no spectacles. However, the third category of iris images was obtained from eight volunteers that used print-patterned contact lenses. Only four images were captured from volunteers in this category as they were not asked to put on spectacles. In addition to the iris images captured, soft biometric features such as age, gender, state of origin, weight, and height of the volunteers were also captured. It is strongly believed that this unique collection of iris datasets of African descent will open up new research in the study of the human iris.",
"keywords": [
"African Human Iris images",
"Age Prediction",
"Ethnicity Prediction",
"Gender Prediction",
"Biometrics",
"Personal Recognition"
],
"content": "Introduction\n\nThe iris recognition system is one of the most widely used and acceptable means of personal recognition and authentication. It has recently become an official means of national identification in India. The Unique Identification Authority of India (UIDAI) has successfully captured 1.5 billion irises from Indian citizens for identification and recognition purposes.1 Many countries have done the same and this increasing popularity and acceptability of human iris as a means of national identification and recognition has called for additional benchmarks, and therefore, new publicly available databases of human iris images.2 Though several human iris datasets exist,3–6 iris datasets of African descents are presently not publicly available. This research effort aimed at bridging this gap in the African continent by embarking on the capture and subsequent creation of human iris images of people of African descent to make them publicly available for research purposes. In the words of Prof. John Daugman:\n\n“There is a more urgent need for an African FACE image database because researchers into face recognition have famously (or infamously) used primarily non-African face images, leading to high levels of bias in algorithms, and disastrous classification performance when they are tested on African face images”.\n\nTherefore, the authors believe the human iris dataset presented in this data article7 will be of great value to researchers willing to advance iris-related research across the African continent. The following are some of the uniqueness of the iris dataset described in this article:\n\n• The dataset presented in this Data Note is the first publicly available human iris dataset of African descent.\n\n• In addition to the iris images, the dataset provides soft biometric features about each volunteer. This additional information will open up new multi-modal biometric research.\n\n• The dataset can serve as a benchmark for evaluating iris recognition methods and other human iris-related research.\n\n• The dataset can be used to validate results obtained from existing iris-related research that used non-African iris images for their research validation.\n\n• The dataset can be used to enhance studies such as personal recognition, age, gender, or ethnicity prediction as well as iris color pigment research on the African continent.\n\n\nMethods\n\nApprovals were obtained from the Ethical and Review Committee of participating Universities before the commencement of the data collection exercise. This was done to ensure and guarantee that the data collection task would not hurt the health of the volunteers and that the publication of the data collected would not infringe on their privacy in any way. Also, all volunteers (willingly without any form of cohesion or pressure) agreed to participate in the iris data collection task with the awareness that the collected data would be made publicly available for research purposes. The privacy of the volunteers was respected as personal details that could make the data traceable to them were not collected.\n\nA Vista EY2H dual iris camera was employed for the iris image capture. The iris camera is a RoHS compliant device that uses a cutting-edge, high-resolution CMOS sensor to produce ISO/IEC 19794-6-compliant images of both irises simultaneously. The camera uses a multi-wavelength near-infrared band of light (NIR: 700nm - 900nm) illumination for superior iris images in all environments. The capturing process meets international eye safety requirements and also has a live eye anti-Spoof detection feature that can be used to reliably detect when a subject is alive. At a click of the capture button, a large 2560 by 720-pixel iris image was produced from the camera; this image is automatically separated into four images with a dimension of 640 by 480 pixels. These are the left and right iris images with the iris region localized, and another set of left and right iris images without the iris section localized. An overview of the camera is provided in Figures 1a-c:\n\nImages have been reproduced from Vista Imaging8 with the appropriate permissions.\n\nDigital Weighing scale: a digital weighing scale as shown in Fig. 2 was employed to measure the weight (soft biometric data) of volunteers.\n\nImage reproduced from www.jumia.com.ng with the appropriate permissions.\n\nDigital Height Measurement Scale: a height measurement scale as shown in Fig. 3 was employed to measure the height of the volunteers.\n\nImage reproduced from www.jumia.com.ng with the appropriate permissions.\n\nThree standard approaches are generally employed for iris image capturing. They are self-enrollment; handheld, self-enrollment (fixed on a tripod); and operator-assisted enrolment. For fast and accurate data capture, the operator-assisted enrolment method was used. The capture device was fixed on a tripod and volunteers were only asked to place their forehead horizontally with their eyes gazing at the lens of the camera. When a perfect range of the irises had been set, the operator clicked the capture button on the camera to initiate the capture process. The captured images were automatically saved on the investigator’s personal computer connected to the scanning device. Afterward, soft biometric features such as: volunteer’s height, weight, age, gender, and state of origin were collected with the respective measuring devices. The data collection sheet shown in Figure 4 was used to initially document the data collected\n\nDetailed information about the iris data collected is presented in this section.\n\nThe human iris images presented in this data article are publicly available on Mendeley Data. The dataset contains 8192 human iris images obtained from 1028 volunteers who were students and members of staff in two Nigerian Universities. The human iris images were captured using a handheld VistaEY2H dual iris camera. The first category of images was captured when the volunteers wore spectacles while the second category of images was captured when the volunteers wore no spectacles. The third category contains iris images captured from volunteers that used print-patterned contact lenses. Moreover, the capture device automatically took four images for each category. All images were saved in .bmp image format. In addition, soft biometrics such as height, weight, age, gender, and state of origin were also collected.\n\n(a) First category of images collected\n\nThe VistaEY2H dual iris camera used for the automatic capture produced four images per volunteer at each capturing instance. These are the right and left iris images of each volunteer as shown in Figure 5, the right and left iris images of each volunteer with the iris region automatically localized; this is shown in Figure 6, the right iris image of the volunteer as shown in Figure 7 and left iris image of the individual as shown in Figure 8.\n\n(b) Second category of images collected\n\nThe second category of images was collected from volunteers that wore spectacles. The images generated were the right and left iris images of each volunteer (as shown in Figure 9), the right and left iris images of each volunteer with the iris region automatically localized (this is shown in Figure 10), right iris image of the volunteer as shown in Figure 11 and left iris image of the volunteer as shown in Figure 12.\n\n(c) Third category of images collected\n\nThe third category was captured from volunteers that used print-patterned contact lenses. Only four images were captured from volunteers in this category as they were not asked to put on spectacles. However, the use of print-patterned contact lenses was not popular among the population considered, therefore, only eight volunteers used lenses. Examples of these images are provided in Figures 13-16.\n\n(d) Soft biometric features\n\nSoft biometric features of each volunteer were also recorded. These are the age, gender, height, weight, and State of origin of volunteers. For instance, the soft biometric features for volunteer B are shown in Table 1:\n\nTo easily distinguish the images, the capturing device automatically generated a unique identification number for each image. For instance, the corresponding unique identification number for each image captured from volunteers A and B are presented in Table 2:\n\nThe supplementary file in Excel format contains the detailed unique identification number for each iris image as well as the biometric features for each volunteer. cell A of the supplementary file is the serial number of the image, cell B contains the last six digits of the unique identification number of a volunteer without spectacles, and cell C contains the last six digits of the unique identification number of the same volunteer with spectacles while cells D to H contain the soft biometric traits of the same volunteer.\n\nA total number of 1028 volunteers participated in the iris image capturing task. The gender of volunteers is provided in Figure 17. The age range of the volunteers are provided in Figure 18.\n\n\nConclusions\n\nThis data article has extensively described the experimental setup behind 1056 human iris datasets of African descent collected to enhance iris-related research in the African continent and beyond. It is believed that the collection could serve as a benchmark for evaluating existing and new iris recognition techniques. Most importantly, the dataset could be used to validate results obtained from existing iris-related research that used non-African iris images for their research validation. The authors look forward to creating more iris datasets captured under different circumstances.",
"appendix": "Data Availability\n\nMendely Data: AFHIRIS: African Human Iris Dataset (Version 1), doi: 10.17632/r3ypmmp2gs.1. 7\n\nMendeley Data: AFHIRIS: African Human Iris Dataset (Version 1) Supplementary File”, doi: 10.17632/gp8vj2379m.1. 9\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).\n\n\nAcknowledgments\n\nThe authors appreciate the Landmark University Centre for Research and Development (LUCRID) for sponsoring the publication of this data article. All volunteers who made the data collection achievable are also appreciated. Most importantly the members of staff and students of Landmark University, Kwara State, Nigeria, and Ladoke Akintola University of Technology Open and Distance Learning Centre, Oyo State, Nigeria.\n\n\nReferences\n\nIndian national identity program tops one billion enrollees:accessed on 4th June 2022.Reference Source\n\nOmelina L, Goga J, Pavlovicova J, et al.: A survey of iris datasets. Image and Vision Computing. 2021; 108: 104109. Publisher Full Text\n\nCASIA Iris Ageing database v. 1.Accessed: 2022-04-01.Reference Source\n\nIIT Delhi Iris Database (Version 1.0).Accessed: 2018-10-24.Reference Source\n\nCUHK Iris Image Dataset.Accessed: 2020-05-20.Reference Source\n\nQuality-Face/Iris Research Ensemble (Q-FIRE).Accessed: 2020-05-20.https://citer.clarkson.edu/researchresources/biometric-dataset-collections-2/quality-faceiris-research-ensemble-qfire/\n\nAkande ON, Ojimba N, Atele O, et al.: AFHIRIS: African Human Iris Dataset (Version 1). Mendeley Data. 2022; V1. Publisher Full Text\n\nVISTAEY2H installation & user manual, version 1.4, Vista Imaging, Inc. http\n\nAkande ON, Ojimba N, Atele O, et al.: AFHIRIS: African Human Iris Dataset (Version 1) Supplementary File. Mendeley Data. 2022; V1. Publisher Full Text"
}
|
[
{
"id": "177096",
"date": "22 Jun 2023",
"name": "Adam Czajka",
"expertise": [
"Reviewer Expertise Biometrics",
"computer vision",
"pattern recognition."
],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nStrengths:\nS1) Relatively large subject pool (1,028 volunteers) compared to other existing iris datasets, significantly increasing the number of iris scans acquired from African subjects available to researchers.\nS2) The dataset is already available and can be downloaded seamlessly. This is highly appreciated.\nS3) Inclusion of the selected demographical information, especially gender.\nS4) Inclusion of subjects wearing glasses and contact lenses.\nSuggestions for improvements:\nI1) The statements that the “Existing publicly available human iris datasets have been collected from non-African subjects therefore, this dataset is the first publicly available human iris dataset of African descent” and that “iris datasets of African descents are presently not publicly available” may be too strong. Iris image datasets collected at universities admitting international students may already include samples from African subjects. For instance, those collected at the University of Notre Dame (https://cvrl.nd.edu/projects/data/) include approx. 1% of iris images acquired from subjects who declared their race as “Black-or-African-American”. There seem to be also one published dataset specifically collected from African subjects: Badejo, Majekodunmi & Atayero (2012)1. I think that listing these past efforts would be worth considering by the authors.\nI2) Probably the most important limitation of this collection is lack of multiple acquisitions from the same eye. This prevents from conducting intra-subject-related research since the genuine comparison scores cannot be generated. I would suggest commenting in the paper the reasons for not taking multiple scans from each eye, since this is rather atypical for biometric data collections.\nI3) Minor suggestions:\nIf the contact lens brands are known, it would be good to add this information to the paper and/or metadata.\n\n“This data article has extensively described the experimental setup behind 1056 human iris datasets of African descent collected to enhance iris-related research in the African continent and beyond.” – Did the authors want to mention “1056 human iris datasets” here?\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": "188975",
"date": "31 Aug 2023",
"name": "J. Jenkin Winston",
"expertise": [
"Reviewer Expertise Biometric recognition and Medical Image 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\nSuggestions for Improvement:\nAlong with the age range of the volunteers, the distribution of gender across each group can also be specified.\n\nThe information on the image pixel resolution of localized irises and left or right iris images can be mentioned to add more information.\n\nThe dataset format of soft biometric details can be mentioned.\n\nThe advantage of Near Infrared Imaging in comparison to Visible Light Imaging can be given to claim the NIR camera modality.\n\n1028 volunteers have participated in image capturing. But, the number of images captured from each subject is missing.\n\nImages with certain artifacts can be included other than spectacles and lens as it can give researchers to try innovative algorithms to work on those biometric images.\n\nCan this dataset be used for liveliness detection?\n\nHistogram of a raw image captured using Vista EY2H can be added.\n\nThe size of the entire dataset can be mentioned with a weblink to access the database.\n\nAn illustrative view on the contained environment used for image capturing can be given.\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/11-1549
|
https://f1000research.com/articles/11-1547/v1
|
21 Dec 22
|
{
"type": "Research Article",
"title": "The Effect of Pyridostigmine as an Antidote for Botulinum Type A: An Experimental Study on Rabbits",
"authors": [
"Huda Salim Alhasan",
"Jawad Hasan",
"Sawsan Alharoon",
"Jawad Hasan",
"Sawsan Alharoon"
],
"abstract": "Background: Botulinum toxin (BoNT) or Botox injections are popular non-surgical and non-invasive option to optimize and change an individual’s facial appearance and achieve rejuvenation. Pyridostigmine is an acetylcholinesterase inhibitor important in clinical practice. This study evaluated the toxic effect of BoNT to demonstrate the efficacy of anticholinesterase inhibitor pyridostigmine. Methods: We conducted an experimental interventional study to evaluate the antidote effect of pyridostigmine against BoNT. Forty rabbits (skeletally mature males, body weight 1000–2000 g) were included in the study. The rabbits were injected with Clostridium botulinum type A neurotoxin complex (BoNT or Botox), which was reconstituted with 0.9% sterile sodium chloride to a concentration of 100 U/2.5 mL. Animals were divided randomly into four groups (10 in each) as follows: Group 1 (control group), group 2 (pyridostigmine-treated group), group 3 (BoNT only group), and group 4 (BoNT + pyridostigmine treated group). Results: In the control group, there was statistical difference between the rabbits’ weight (P=0.03). The left muscle was significantly heavier than the right (P=0.025). In the pyridostigmine only group, the rabbits’ weight had a high statistical difference during four weeks of the study (P=0.002). In the BoNT injection group, weight had high statistically significant difference four weeks of the study (P=0.033), which was more in first week (1.55±0.2 kg) and then subsequently decreased in the fourth week (1.345±0.13 kg). The mean weight of the rabbits’ right quadriceps muscle was 6.573±1.3 g, which was significantly lower than the mean weight of the rabbits’ left quadriceps muscle (8.09±1.2 g, P=0.0001).\nConclusions: To the best of our knowledge, this is the first study conducted in Iraq to investigate the antidote effect of pyridostigmine against BoNT injections. Pyridostigmine and saline caused increased weight of rabbits in comparison to rabbits that received BoNT injections. Pyridostigmine can act as strong antidote against Botox toxicity.",
"keywords": [
"BOTOX injections",
"Pyridostigmine",
"neurotoxin",
"clostridium botulinum type-A"
],
"content": "Introduction\n\nAcetylcholinesterase inhibitors, such as pyridostigmine, are important in clinical practice, i.e., they can be used to treat the symptoms associated with myasthenia gravis, Alzheimer’s disease, and multiple sclerosis.1–3 Acute or prolonged exposure to reversible acetylcholinesterase inhibitors in healthy individuals, however, can have positive or negative effects on neuromuscular transmission and muscle ultrastructure.4,5 Classically, the theory of receptor control suggests that chronic agonist stimulation of any receptor can lead to receptor desensitization and even down-regulation in a concentration- and time-dependent manner.6–9\n\nClostridium botulinum is a rod-shaped, gram-positive anaerobic bacterium. It is a ubiquitous soil microorganism that can remain dormant as a spore in favorable environments. Its spores can germinate and produce a neurotoxin known as botulinum toxin.10 In 1897, Van Ermengem discovered this bacterium when Belgian patients presented with botulism. After that, several strains of the bacterium that produced serologically distinct toxin types were identified and classified into seven serotypes, A, B, C1, D, E, F, and G.11 In 1928, Dr. Herman Sommer isolated the most potent serotype, botulinum neurotoxin (BoNT) type A.12 Each serotype has its own distinct pharmacological properties,13,14 and only materials containing BoNT type A or type B have been approved for human use. These neurotoxins act on the peripheral nervous system, where they inhibit the release of acetylcholine (ACh) from the synaptic terminal of neuromuscular junctions.15,16 Currently, BoNT type A injections are a popular non-surgical and non-invasive treatment to optimize and change an individual’s facial appearance and achieve rejuvenation.17\n\nIn this study, we aimed to demonstrate the efficacy of anticholinesterase inhibitor pyridostigmine in accelerating spontaneous recovery following botulinum toxin injections in a rabbit model.\n\n\nMethods\n\nAn experimental interventional study was conducted on 40 rabbits to evaluate the antidote effect of pyridostigmine against BoNT. This study was one-way one-period (28 days) from 12th May to 11th June 2022. Rabbits were randomly divided into four groups, and each group consisted of 10 rabbits. Control group received distal water, pyridostigmine-treated group received oral pyridostigmine, BoNT only group received injections intramuscularly with a single dose of BoNT and BoNT+pyridostigmine treated group received injections with BoNT and oral pyridostigmine, as shown in the flow chart (Figure 1).\n\nThe drugs and their suppliers used in the study are listed in Table 1.\n\nThe drugs were bought from private pharmacies in Basrah city, Iraq. Tablets were crushed using a mortar and pestle, and then dissolved in distilled water to obtain a solution for administration to the rabbits. The doses of each drug administered were prepared as follows.\n\nOne 60 mg tablet of pyridostigmine was dissolved in 50 mL distilled water before each administration to obtain a suspension of concentration 1.2 mg/mL. The powder was completely dissolved to give a homogenous solution in a sealed glass container. A dose of 4 mL/kg from the prepared stock solution was orally administered to each rabbit according to their body weight for an accurate dose (5 mg/kg), and then the dose was increased to 7 mg/kg after three days to avoid any unwanted cholinergic effects.\n\nUsing an appropriate-sized needle and syringe, 2.5 mL of 0.9% non-preserved sterile saline was drawn up. The needle was inserted and the saline was slowly injected into the vial. A vacuum was present in the vial, which demonstrated that it was sterile. The syringe was then disconnected from the needle and the BoNT was gently mixed with the saline by rotating the vial. The date and time of reconstitution were recorded on the label. After that, a new sterile syringe was attached and the reconstituted fluid was drawn into the syringe by angling the needle into the bottom corner of the vial to enable complete extraction. Any air bubbles in the syringe were expelled. The syringe was then disconnected from the needle used for reconstitution and a 30-gauge needle was attached for the injection. The doses of the drugs used in this study were selected according to a literature review and a pilot study,18 as follows: Pyridostigmine19 and BoNT.20\n\nThe sample size calculation of this study is based on availability of rabbit, effect of group size on the findings, and significant of this number on the experimental level.\n\nInclusion criteria\n\nForty rabbits (Oryctolagus cuniculus) (skeletally mature males) were included in the study aged from 2–4 years. They were obtained from a local market in Basrah city, and their body weights were between 1000 and 2000 g. One animal died during the study period. The rabbits were locally bred and sexually mature. Animals were housed under a controlled animal house conditioned atmosphere at a constant temperature (25°C±3) and relative humidity (50±5%) and supplied with food and water ad libitum during the study period. Animals were kept under observation for one month and fasted for three hours at the time of dosing.\n\nExclusion criteria\n\nRabbits were excluded from the study when dying, and when any adverse effect seen after administration of drugs like bleeding at site of injection, vomiting and cramping.\n\nRabbits were acclimatised for seven days before the study. They were allowed normal cage (65 × 345 × 330 cm3) activity and fed a standard diet (refoil and lettuce). To reduce stress, rough handling and overcrowding were avoided. During the four weeks of treatment, the body weight of each animal was recorded weekly. All authors were aware of groups during allocation, at a period of conducting the experiment, at the time of the outcomes assessment and at the time of analysis of data.\n\nThis study was approved by the Ethical Committee of the Council of College of Medicine at the University of Basrah (No. 4502).\n\nThe animals were randomly divided into four groups (10 rabbits in each group). Each group was treated for four weeks as follows (Table 2):\n\n• Group 1 (control group)\n\nRabbits in this group were treated with 4 mL/kg distilled water orally for three days. Then the dose was increased to 6 mL/kg and they were injected with distilled water (10 U/kg) in the right quadriceps muscle as a single dose and euthanized on day 30 of the study.\n\n• Group 2 (pyridostigmine-treated group)\n\nRabbits in this group were orally administered pyridostigmine (4 ml/kg). Then the dose was increased to 6 mL/kg for the remaining 30 days. The rabbits were euthanized at the end of the study.\n\n• Group 3 (BoNT only group)\n\nRabbits in this group were intramuscularly injected with a single dose of BoNT (10 U/kg) in the right quadriceps muscle (targeting the rectus femoris, vastus lateralis, vastus intermedius, and the vastus medialis) and euthanized on day 30 of the study.\n\n• Group 4 (BoNT+pyridostigmine treated group)\n\nRabbits in this group were intramuscularly injected with BoNT (10 U/kg) into the right quadriceps muscle (single dose) and orally administered pyridostigmine (4 ml/kg) for three days. The dose was then increased to 6 mL/kg for the remaining 30 days before the rabbits were euthanized at the end of the study.\n\nThe following items were assessed: rabbits weight (kg) at the beginning of the study and at end of each of subsequent week, and weight of rabbit quadriceps muscle (g) after the end of the study.\n\nThe statistical analysis was performed using SPSS version 20 (IBM Inc., Chicago, IL, USA), Resource Identification Portal (RRID:SCR_004098).Descriptive statistics, consisting of numbers and percentages, are provided. The mean, median, range, minimum, maximum, and standard deviation (SD) were calculated for categorical data. Associations between groups were assessed by an unpaired t-test. Associations between variables were assessed by a chi-squared test. An ANOVA was used to describe associations between groups. A two-sided P-value of less than 0.05 was considered statistically significant. Details can be viewed at https://scicrunch.org/resources/data/record/nlx_144509-1/SCR_004098/resolver?q=SPSS%20version%2020&l=SPSS%20version%2020&i=rrid:scr_004098.\n\n\nResults\n\nFor the control group, the rabbits’ weight during one month of the experiment is shown in Table 3 and Figure 2. There was a statistically significant difference of weights during four weeks in the control group (P=0.03), which was more being in the third week (mean±SD=1.5±0.122 kg).\n\nThe mean weight of the rabbits on the first day was 1.415±0.18 kg, whereas the mean weight of rabbits at the end of the fourth week was 1.475±0.145 kg, after one month of 0.9% saline injection in the control group, as shown in Table 4. There was no significant difference between groups (t=2.092, 95% CI=0.125–0.005, P=0.066).\n\nIn this study, the mean weight of the rabbits’ right quadriceps muscle was 8.28±1.06 g, while the mean weight of the rabbits’ left quadriceps muscle was 8.385±1.06 g. The left muscle was significantly heavier than the right (t=2.677, 95%CI=0.193–0.016, P=0.025), as shown in Table 5 and Figure 3.\n\nFor the pyridostigmine-only group, the rabbits’ weight during one month of the study is shown in Table 6 and Figure 4. There was a high statistically significant difference among rabbits weight during four weeks of the study in this group (P=0.002), which was more being in the third week (mean±SD=1.485±0.175 kg).\n\nIn the pyridostigmine-only group, the mean weight of rabbits on the first day was 1.45±0.227 kg, which was lower than the mean weight of the rabbits at the end of the fourth week, which was 1.475±0.178 kg after one month of oral pyridostigmine, as shown in Table 7. There was no significant difference between groups (t=1.103, 95%CI=0.076–0.026, P=0.299).\n\nIn the pyridostigmine-only group, the mean weight of the rabbits’ right quadriceps muscle was 8.73±1.23 g, which was higher than the mean weight of the rabbits’ left quadriceps muscle, which was 8.635±1.14 g, but the difference was not significant (t=1.57, 95% CI=-0.042– ‐0.232, P=0.151), as shown in Table 8 and Figure 5.\n\nFor the BoNT injection group, the rabbits’ weight during one month of the study is shown in Table 9 and Figure 6. There was a high statistically significant difference among rabbits weight during four weeks of the study in this group (P=0.033), which was more being in the first week (mean±SD=1.55±0.2 kg) but then dropped subsequently in the fourth week (mean±SD=1.345±0.13 kg).\n\nThe mean weight of the rabbits on the first day was 1.55±0.2 kg, which was higher than the mean weight of the rabbits at the end of the fourth week, which was 1.345±0.13 kg, after one month of BoNT injections, as shown in Table 10. There was a highly significant difference between groups (t=5.156, 95% CI=0.115–0.295, P=0.001).\n\nIn the Botox group, the mean weight of the rabbits’ right quadriceps muscle was 6.573±1.3 g, which was lower than the mean weight of the rabbits’ left quadriceps muscle, which was 8.09±1.2 g, a highly significant difference (t=21.795, 95% CI=1.674–1.36, P=0.0001), as shown in Table 11 and Figure 7.\n\nFor the pyridostigmine and BoNT injection group, the rabbits’ weight during one month of the study is shown in Table 12 and Figure 8. There was a statistically significant difference among rabbits’ weight during four weeks of the study (P=0.027), which was more being in the first week (mean±SD=1.455±0.2 kg).\n\nThus, the mean weight of the rabbits on the first day was 1.455±0.2 kg, which was the same as the mean weight of rabbits at the end of the fourth week, which was 1.455±0.18 kg, after one month of BoNT injection, as shown in Table 13. There were no significant differences between the groups (t=0, 95% CI=N/A, P=1).\n\nIn this study, the mean weight of the rabbits’ right quadriceps muscle was 5.564±0.615 g, which was lower than the mean weight of the rabbits’ left quadriceps muscle, which was 8.36±0.55 g, a highly statistically significant difference (t=18.656, 95% CI=3.135–2.457, P=0.0001), as shown in Table 14 and Figure 9.\n\nTable 15 and Figure 10 show the ANOVA analysis and linear correlation among different groups of the study in relation to the control group in the first week. There was a significant relationship between group A and B in the first week (P=0.024). There were no associations between group A and the other categories.\n\nTable 16 and Figure 11 show the ANOVA analysis and linear correlation among different groups of the study in relation to the control group in the second week. There was a significant relationship between group A and D in the second week (P=0.047).\n\nTable 17 and Figure 12 show the ANOVA analysis and linear correlation among different groups of the study in relation to the control group in the third week. There was a significant relationship between group A and B in the third week (P=0.015).\n\nTable 18 and Figure 13 show the ANOVA analysis and linear correlation among different groups of the study in relation to the control group in the fourth week. There was no significant difference between group A and the other groups.\n\nFigure 14 shows the correlation among different groups of the study in relation to the rabbits’ right quadriceps muscle weight. There was a highly statistically significant difference among groups according to the ANOVA analysis (F=18.515, P<0.0001).\n\nFigure 15 shows the correlation among different groups of the study in relation to the rabbits’ left quadriceps muscle weight. There was no statistically significant difference among groups according to the ANOVA analysis (F=0.475, P=0.7).\n\n\nDiscussion\n\nRecently, a model for BoNT intoxication was developed in rabbits for the first time.21 This is in accordance with the animal rule of the US FDA.22 Rabbits are the third most commonly used species for experimental research in the western countries.23 Rabbits are larger than mice, so blood sampling and intravenous administration of drugs are easy.24 Phylogenetically, rabbits are closer to humans than rodents, and due to their anatomical, physiological, genetic, and biochemical similarities, they are used as animal models for human diseases in a variety of medical research fields.25\n\nIn this series, among the control group, the rabbits weight increased, reaching peak weight in the third week, with a statistically significant difference (P=0.03). The mean weight of rabbits on the first day was 1.415±0.18 kg, whereas at the end of the fourth week it was 1.475±0.145 kg, without significant difference between groups (P=0.066). Furthermore, the mean weight of the rabbits’ right quadriceps muscle was lower than the mean weight of the rabbits’ left quadriceps muscle, with a highly statistically significant difference (P=0.025). This is the same as found in Morsch et al.’s,26 experiment, except they used citric acid monohydrate, sodium citrate dehydrate, methyl paraben, propyl paraben, and NaCl in sterile water (pH 5.1). However, in the saline group of Richtsfeld et al.,27 there were no such changes, whereas an agreement was seen in saline injected group that showed gradual increased in the body mass of animals.28\n\nIn the pyridostigmine only group, the weight during the study peaked in the third week, with a highly statistically significant difference (P=0.002). The mean weight of rabbits on the first day was lower than the mean weight of rabbits at the end of the fourth week. The mean weight of the rabbits’ right quadriceps muscle was higher than the mean weight of the rabbits’ left quadriceps muscle, with no significant difference (P=0.151). This agrees with the findings of Morsch et al.26 They delivered pyridostigmine to animals systemically and continuously for seven to nine days via a minipump. Haigh et al. found that the effect of different doses of pyridostigmine on whole-blood acetylcholinesterase activity was recorded seven days after ingestion.29 Morsch et al.26 found rabbits treated with pyridostigmine lost weight and developed severe muscle weakness within two weeks. Hence, rather than preventing muscle weakness, pyridostigmine was capable of precipitating this weakness. This is in disagreement with what was reported by Richtsfeld et al., who found that animals in the pyridostigmine group lost 10% of their initial body weight during administration of pyridostigmine.27\n\nMorsch et al. concluded that pyridostigmine remains the only approved symptomatic drug for the treatment of BoNT intoxication, and one week of ingestion was enough to exacerbate neuromuscular impairment.26\n\nIn the BoNT injection group, the rabbits’ weight was more in the first week (mean±SD=1.55±0.2 kg) but then subsequently decreased by the fourth week (mean±SD=1.345±0.13 kg); this was a highly statistically significant difference (P=0.033). Although, the mean weight on the first day (1.55±0.2 kg) was higher than the mean weight at the end of the fourth week (1.345±0.13 kg) of the study; this was a high significant difference (P=0.001). The mean weight of the rabbits’ right quadriceps muscle was 6.573±1.3 g, which was lower than the mean weight of the left quadriceps muscle (8.09±1.2 g), with a highly significant difference (P=0.0001). In the pyridostigmine and BoNT injection group, the rabbits’ weight gradually declined during subsequent injections. There was a statistically significant difference between the rabbits’ weights (P=0.027). Thus, the mean weight of rabbits on the first day was 1.455±0.2 kg, which had decreased at the end of the fourth week to 1.455±0.18 kg, with no significant between groups (t=0, 95% CI=N/A, P=1). In addition, the mean weight of the rabbits’ right quadriceps muscle was lower than the left quadriceps muscle, with a highly statistically significant difference (P=0.0001). Similarly, the animals lost weight and became weak after two weeks in Morsch et al.’s study.26 Also, our study’s results are consistent with previous experiments,30 and an agreement has been seen in Botox mice by Warner et al.28\n\nTorgeman et al. used different symptomatic rabbits dosing group with increased in a dose-dependent manner: 50%, 75%, and 80% with toxin doses of 0.5 ng/kg, 0.65 ng/kg, and 0.75 ng/kg, respectively. They found that 100% of the rabbits became symptomatic only in the 0.85 ng/kg dose, despite its lethal exposure dose.21\n\nIn our ANOVA analysis of this study, we showed the following findings between the control group and the other groups, such as a significant relationship between group A and B in the first week (P=0.024). There was a significant relationship between group A and D in the second week (P=0.047). There was a significant relationship between group A and B in the third week (P=0.015). The correlation among different groups in relation to the rabbits’ right quadriceps muscle weights had a highly statistically significant difference among groups according to the ANOVA analysis (P<0.0001). However, there was no statistically significant difference in the correlation with the rabbits’ left quadriceps muscle weight (P=0.7). Similarly, there were significant differences in the weights of the tibialis muscle between groups in different study periods.27\n\nIn mice series, BoNT treatment significantly diminished right hindlimb muscle mass in both the quadriceps (102.9±31.2 vs. 195.3±13.9 mg, −47.3%, P<0.001) and calf (58.4±13.3 vs. 145.0±3.9 mg, −59.7%, P<0.001) compared to saline mice. Muscle mass in the contralateral hindlimb (left) of the BoNT-treated mice was also significantly diminished compared with that of the saline-treated mice, but to a lesser extent (quadriceps: 166.0±5.9 vs. 199.9±2.7 mg, minus 17.0%; calf: 120.8±4.2 vs. 145.0±1.6 mg, minus 16.7%, both P<0.001),28 which seem similar to the current study’s findings.\n\nIncreasing evidence indicates that prolonged exposure to pyridostigmine was an etiological factor for Gulf War syndrome, which includes skeletal muscle symptoms.31 This could be explained by the fact that certain muscles appear to be affected more than others, for reasons unknown, but possibly due to quantitative differences in etiology.30,32,33\n\n\nConclusions\n\nTo the best of our knowledge, this is the first study conducted in Iraq to investigate the antidote effect of pyridostigmine against BoNT injections. Pyridostigmine and saline caused increased weight in rabbits in comparison to BoNT-injected rabbits. No significant effect was seen on muscles after pyridostigmine administration, while the inverse was observed in the BoNT group. Pyridostigmine can act as a strong antidote against BoNT intoxication.",
"appendix": "Data availability\n\nAlhasan Huda Salim, Jawad K. Hasan, & Sawsan S. Al-Haroon. (2022). The effect of Pyridostigmine As Antidote for Botulinum type A. https://doi.org/10.5281/zenodo.7250444. 34\n\nThe ARRIVE guidelines 2.0: author https://arriveguidelines.org/sites/arrive/files/documents/Author%20Checklist%20-%20Full.pdf\n\nHuda Salim, Jawad Hasan, & Sawsan Alharoon. (2022). ARRIVE checklist of Pyridostigmine and BoNT. Zenodo. https://doi.org/10.5281/zenodo.7362881. 35\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\nArgov Z: Management of myasthenic conditions: nonimmune issues. Curr. Opin. Neurol. 2009; 22(5): 493–497. PubMed Abstract | Publisher Full Text\n\nDarreh-Shori T, Soininen H: Effects of cholinesterase inhibitors on the activities and protein levels of cholinesterases in the cerebrospinal fluid of patients with Alzheimer's disease: a review of recent clinical studies. Curr. Alzheimer Res. 2010; 7(1): 67–73. PubMed Abstract | Publisher Full Text\n\nLyros E, Messinis L, Papageorgiou SG, et al.: Cognitive dysfunction in multiple sclerosis: the effect of pharmacological interventions. Int. Rev. Psychiatry. 2010; 22(1): 35–42. Publisher Full Text\n\nKitajima O, Suzuki T, Fukano N, et al.: Masui. Jpn. J. Anesthesiol. 2009; 58(4): 410–415. PubMed Abstract\n\nHerbstreit F, Zigrahn D, Ochterbeck C, et al.: Neostigmine/glycopyrrolate administered after recovery from neuromuscular block increases upper airway collapsibility by decreasing genioglossus muscle activity in response to negative pharyngeal pressure. Anesthesiology. 2010; 113(6): 1280–1288. PubMed Abstract | Publisher Full Text\n\nBinyaminy B, Gafni M, Shapira M, et al.: Agonist-specific down regulation of mu-opioid receptors: Different cellular pathways are activated by different opioid agonists. Life Sci. 2008; 82(15–16): 831–839. PubMed Abstract | Publisher Full Text\n\nMauck B, Lucot JB, Paton S, et al.: Cholinesterase inhibitors and stress: effects on brain muscarinic receptor density in mice. Neurotoxicology. 2010; 31(5): 461–467. PubMed Abstract | Publisher Full Text\n\nRole LW, Talmage DA: Neurobiology: new order for thought disorders. Nature. 2007; 448(7151): 263–265. Publisher Full Text\n\nOsadchii OE, Norton GR, McKechnie R, et al.: Cardiac dilatation and pump dysfunction without intrinsic myocardial systolic failure following chronic beta-adrenoreceptor activation. Am. J. Physiol. Heart Circ. Physiol. 2007; 292(4): H1898–H1905. PubMed Abstract | Publisher Full Text\n\nSobel J, Tucker N, Sulka A, et al.: Foodborne botulism in the United States, 1990-2000. Emerg. Infect. Dis. 2004; 10(9): 1606–1611. PubMed Abstract | Publisher Full Text | Free Full Text\n\nGallagher CJ, Ackerman A:History of Botulinum Toxin for Medical and Aesthetic Use. Botulinum Toxins: Cosmetic and Clinical Applications. First Edition.Cohen JL, Ozog DM, editors.© 2017 JohnWiley & Sons Ltd;2017; pp: 37–45. Published 2017 by JohnWiley & Sons Ltd. Publisher Full Text\n\nCarruthers A, Carruthers J:History of Botulinum Toxin for Medical and Aesthetic Use. Botulinum Toxins: Cosmetic and Clinical Applications. First Edition. Cohen JL, Ozog DM, editors.© 2017 JohnWiley & Sons Ltd;2017; pp: 1–7. Published 2017 by JohnWiley & Sons Ltd. Publisher Full Text\n\nComella CL, Jankovic J, Shannon KM, et al.: Comparison of botulinum toxin serotypes A and B for the treatment of cervical dystonia. Neurology. 2005; 65(9): 1423–1429. PubMed Abstract | Publisher Full Text\n\nAoki KR: A comparison of the safety margins of botulinum neurotoxin serotypes A, B, and F in mice. Toxicon. 2001; 39(12): 1815–1820. PubMed Abstract | Publisher Full Text\n\nAlshadwi A, Nadershah M, Osborn T: Therapeutic applications of botulinum neurotoxins in head and neck disorders. Saudi Dent. J. 2015; 27(1): 3–11. PubMed Abstract | Publisher Full Text | Free Full Text\n\nHyodo M, Hirose K, Nagao A, et al.: Botulinum Toxin Therapy for Spasmodic Dysphonia in Japan: The History and an Update. Toxins. 2022; 14(7): 451. PubMed Abstract | Publisher Full Text | Free Full Text\n\nStatista: U.S. patient satisfaction: top nonsurgical cosmetic procedures 2019.Accessed April 30, 2022.Reference Source\n\nCai BB, Francis J, Brin MF, et al.: Botulinum neurotoxin type A-cleaved SNAP25 is confined to primary motor neurons and localized on the plasma membrane following intramuscular toxin injection. Neuroscience. 2017; 352: 155–169. PubMed Abstract | Publisher Full Text\n\nKluwe WM, Page JG, Toft JD, et al.: Pharmacological and toxicological evaluation of orally administered pyridostigmine in dogs. Fundam. Appl. Toxicol. 1990; 14(1): 40–53. Publisher Full Text\n\nPingel J, Nielsen MS, Lauridsen T, et al.: Injection of high dose botulinum-toxin A leads to impaired skeletal muscle function and damage of the fibrilar and non-fibrilar structures. Sci. Rep. 2017; 7(1): 14746. PubMed Abstract | Publisher Full Text | Free Full Text\n\nTorgeman A, Diamant E, Dor E, et al.: A Rabbit Model for the Evaluation of Drugs for Treating the Chronic Phase of Botulism. Toxins. 2021; 13(10): 679. PubMed Abstract | Publisher Full Text | Free Full Text\n\nFDA: Approved Products: Botox (onabotulinumtoxinA) for injection.2021.Reference Source\n\nHedenqvist P, Edner A, Fahlman Å, et al.: Continuous intravenous anaesthesia with sufentanil and midazolam in medetomidine premedicated New Zealand White rabbits. BMC Vet. Res. 2013; 9: 21. PubMed Abstract | Publisher Full Text | Free Full Text\n\nWeber K, Mowat V, Hartmann E, et al.: Pathology in Continuous Infusion Studies in Rodents and Non-Rodents and ITO (Infusion Technology Organisation)-Recommended Protocol for Tissue Sampling and Terminology for Procedure-Related Lesions. J. Toxicol. Pathol. 2011; 24(2): 113–124. PubMed Abstract | Publisher Full Text | Free Full Text\n\nKamaruzaman NA, Kardia E, Kamaldin N, et al.: The rabbit as a model for studying lung disease and stem cell therapy. Biomed. Res. Int. 2013; 2013: 691830.\n\nMorsch M, Reddel SW, Ghazanfari N, et al.: Pyridostigmine but not 3,4-diaminopyridine exacerbates ACh receptor loss and myasthenia induced in mice by muscle-specific kinase autoantibody. J. Physiol. 2013; 591(10): 2747–2762. PubMed Abstract | Publisher Full Text | Free Full Text\n\nRichtsfeld M, Yasuhara S, Fink H, et al.: Prolonged administration of pyridostigmine impairs neuromuscular function with and without down-regulation of acetylcholine receptors. Anesthesiology. 2013; 119(2): 412–421. PubMed Abstract | Publisher Full Text\n\nWarner SE, Sanford DA, Becker BA, et al.: Botox induced muscle paralysis rapidly degrades bone. Bone. 2006; 38(2): 257–264. PubMed Abstract | Publisher Full Text | Free Full Text\n\nHaigh JR, Lefkowitz LJ, Capacio BR, et al.: Advantages of the WRAIR whole blood cholinesterase assay: comparative analysis to the micro-Ellman, Test-mate ChE, and Michel (DeltapH) assays. Chem. Biol. Interact. 2008; 175(1-3): 417–420. PubMed Abstract | Publisher Full Text\n\nCole RN, Ghazanfari N, Ngo ST, et al.: Patient autoantibodies deplete postsynaptic muscle-specific kinase leading to disassembly of the ACh receptor scaffold and myasthenia gravis in mice. J. Physiol. 2010; 588(Pt 17): 3217–3229. PubMed Abstract | Publisher Full Text | Free Full Text\n\nLucas KE, Rowe PC, Armenian HK: Latency and exposure-health associations in Gulf War veterans with early fatigue onsets: a case-control study. Ann. Epidemiol. 2007; 17(10): 799–806. PubMed Abstract | Publisher Full Text\n\nXu K, Jha S, Hoch W, et al.: Delayed synapsing muscles are more severely affected in an experimental model of MuSK-induced myasthenia gravis. Neuroscience. 2006; 143(3): 655–659. PubMed Abstract | Publisher Full Text\n\nPunga AR, Lin S, Oliveri F, et al.: Muscle-selective synaptic disassembly and reorganization in MuSK antibody positive MG mice. Exp. Neurol. 2011; 230(2): 207–217. PubMed Abstract | Publisher Full Text\n\nSalim AH, Hasan JK, Al-Haroon SS: The effect of Pyridostigmine As Antidote for Botulinum type A.2022. Publisher Full Text\n\nSalim H, Hasan J, Alharoon S: ARRIVE checklist of Pyridostigmine and BoNT. Zenodo.2022. Publisher Full Text"
}
|
[
{
"id": "169144",
"date": "26 Apr 2023",
"name": "Christine Rasetti-Escargueil",
"expertise": [
"Reviewer Expertise Pharmacology",
"neurotoxins",
"botulinum",
"antitoxins",
"ex vivo",
"in vivo",
"in vitro models",
"stem cells."
],
"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\nAbstract: -------------\nThe \"Botox\" is not the only approved medicine containing Botulinum toxin. Please remove \"Botox\" or add: \"Botox like\" products.\nPlease state briefly the main uses of pyridostigmine in the clinic.\nPlease modify the third sentence to: \"This study evaluated the efficacy of pyridostigmine against toxic effects of BoNT\".\nResults: Please state if differences in rabbit weight was between controls and treated or within the control group?\nPlease state what difference in weight occurred in the pyridostigmine only group, also for the Botox group: please only state that there was a statistically significant decrease of weight after 4 weeks of treatment.\nIntroduction: ------------------\nPlease detail BoNT mechanism of action and how the pyridostigmine could counteract the mechanism of BoNT effects on the neuromuscular junction.\nAlso, please give more details on the therapeutic uses of BoNT. There are a lot of diverse therapeutic indications.\nMethods: -------------\nFlow chart: the control group should receive saline solution, not distilled water. This is not recommended for animal welfare. Did you see any detrimental effects (pain) during water injections?\nDrugs: Please explain how the Units were calculated for the Botulinum toxin A (Hutox).\nPlease indicate Hutox instead of Botox since Hutox was used for reconstitution (not the product Botox). Only sterile saline should be used for injection into the rabbits due to infection risks.\nSample size: please clarify the sentence: \"significant of this number on the experimental level\".\nInclusion criteria: the rabbits should be obtained from an approved supplier to avoid risks of disease in the rabbits that can influence the results of the study.\nExclusion criteria: The death, vomiting or cramping should not justify exclusion from the study since they are probably due to the treatments: Hutox or pyridostigmine.\nIt is important to take into account all the effects to assess true efficacy.\nOutcome: Other typical symptoms of botulism should be assessed like altered breathing, weakness, paralysis or vomiting.\nResults: ------------ Control group Table 4: please clarify if the saline solution or distilled water was injected into rabbits of the control group?\nFigure 3: considering the high variability of the difference in quadriceps weight, this is unlikely to be significant.\nPyridostigmine group: Please clarify the sentence: \"In the pyridostigmine-only group, the mean weight of the rabbits’ right quadriceps muscle was higher than the mean weight of the rabbits’ left quadriceps muscle (8.73±1.23 g for the right quadriceps versus 8.635±1.14 g for the left quadriceps),\"\n\nBoNT group: Please state: \"There was a dramatic decrease in rabbit weight after BoNT injections during the four weeks of the study. The decrease was highly significant.\"\n\"Similarly, there was a significant decrease of the right quadriceps muscle that was injected with BoNT, compared to the left muscle (not injected).\"\nIt would be good to describe symptoms following BoNT injection and compare with other treated groups and control group.\nPyridostigmine and BoNT injection group It is unexpected to see a statistically significant difference among rabbits’ weight during four weeks of the study considering the level of variability (0.2kg) compared to the very small differences (1.455kg versus 1.42kg: 0.035kg).\nComments for the Box plots of rabbit quadriceps weight: please remove \"injected\" for the left muscles as only right muscles were treated.\nANOVA analysis\nPlease provide detailed analysis of the ANOVA analysis specifically regarding the drop-line correlation of rabbit weight for each week as it is unclear at present how to interpret the drop-line plots. Please provide a definition of x and y axis for this type of plots.\nDiscussion ----------------- This is not the first paper describing a rabbit model. Please see for example: \"A Novel Rabbit Spirometry Model of Type E Botulism and Its Use for the Evaluation of Post-symptom Antitoxin Efficacy\"; February 2018; Antimicrobial Agents and Chemotherapy 62(4):AAC.02379-17 DOI:10.1128/AAC.02379-17.\nPlease explain why the right quadriceps muscle is smaller than the left muscle in the control group.\nPlease correct increased with \"increase\" in: \"whereas an agreement was seen in saline injected group that showed gradual increased in the body mass of animals.28\"\nPlease explain why Morsh et al. found different effects of pyridostigmine on rabbit weight.\nPlease clarify the sentence for BoNT group: \"In the BoNT injection group, the rabbits’ weight significantly decreased by the fourth week (in the first week: mean±SD=1.55±0.2 kg versus mean±SD=1.345±0.13 kg on the fourth week); this was a highly statistically significant difference (P=0.033)\".\n\nThis sentence is not in line with the data of BoNT group: \"Thus, the mean weight of rabbits on the first day was 1.455±0.2 kg, which had decreased at the end of the fourth week to 1.455±0.18 kg, with no significant between groups (t=0, 95% CI=N/A, P=1). \"\n\"Torgeman et al. used different symptomatic rabbits dosing group with increase in a dose-dependent manner\".\nThe Anova analysis deserve more explanations as it is not clear at present for non statisticians. Please detail how the correlations were done and which parameters were 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? 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": []
},
{
"id": "190325",
"date": "17 Aug 2023",
"name": "Lidija Bach-Rojecky",
"expertise": [
"Reviewer Expertise Neuropharmacology",
"neuroscience",
"pain research",
"botulinum toxin investigation"
],
"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\nAbstract:\nBOTOX is not the only botulinum toxin type A product, so it should be deleted from the first sentence\n\nMethods: It should be stated how the toxin was injected (site) and what amount (dose and volume) of the toxin was injected.\n\nResults: The results presentation is unclear and confusing.\n\nConclusion: The sentence “Pyridostigmine and saline caused the increased weight of rabbits in comparison to rabbits that received BoNT“ should be changed to „Pyridostigmine and saline didn't affect weight gain of rabbits in contrast to BoNT treated group“\n\nFrom these results certainly cannot be concluded that „Pyridostigmine can act as a strong antidote against Botox toxicity“.\n\nKeywords: delete „Botox injections“ and neurotoxin; add: rabbit, antidote\nIntroduction:\nComment to sentence: “Acetylcholinesterase inhibitors, such as pyridostigmine, are important in clinical practice, i.e., they can be used to treat the symptoms associated with myasthenia gravis, Alzheimer’s disease, and multiple sclerosis“ - Pyridostigmine due to its structure does not cross the blood-brain barrier and is not used in Alzheimer disease or for any cognitive dysfunction.\n\nThe mechanism of BoNT action should be described\n\nBoNT has many various indications that should be mentioned\nMethods:\nThis section needs significant editing and improvement\n\nAnimals should have been purchased from the registered breeder-supplier\n\nThe sex of the rabbits and method of randomization should be stated; persons involved in performing experiments and data analysis were not blinded to treatments\n\nTable 1 – atropin sulfate was not used in the experiments and is mentioned in the table\n\nTable 1 – column related to the potency of BoNT – 100 Units is contained in one vial; It is important to know the quantity of botulinum toxin that corresponds to 1 U. Is the titer of 1 U HUTOX equal to that of 1 U BOTOX?\n\nThe method of euthanasia is not stated\n\nHow was oral administration performed?\n\nWere the animals inspected for signs of suffering and deteriorated health status?\n\nFor the control group the authors state that distilled water (10 U/kg) was injected. 10U/kg should be deleted (the same refers to Table 2)\n\nOutcomes: It is a pity that different parameters and signs of potential toxicity were not examined. Weight loss is the only sign of systemic toxicity that was checked (respiration symptoms, salivary secretion, etc.), while muscle weight loss is a direct consequence of peripheral BoNT action in neuromuscular junctions.\nResults:\nThis section needs complete re-structure and rewriting.\n\nThe results should be presented to avoid repetition of the same data in tables and figures. Also, data (for rabbits’ weight and muscle weights) for all experimental groups should be presented in one figure for easier comparison of the results.\n\nIt is unusual and unnecessary to present data for every experimental group in a separate table/figure. The results are shown in 15 figures and 18 tables, which is too much and hard to follow.\n\nAlso, the textual description of the results is confusing, For example, the authors state that “There was no significant difference between groups“ when describing one concrete experimental group (i.e. control group). I suppose the authors mean that there was no difference between the data at different time points.\n\nResults of ANOVA analysis should be described in more detail.\nDiscussion:\nThe second, third, and fourth sentences are not relevant to the present study. They could be omitted.\n\nResults are once again repeated in the discussion section; there is no need to repeat the same sentences from the results section\n\nThe sentence \"However, in the saline group of Richtsfeld et al (27) there were no such changes, whereas an agreement was seen in the saline-injected group that showed a gradual increase in the body mass of animals (28)“ is unclear and needs rewriting. The authors should be careful regarding the citation of other authors' studies and be clear about the interpretation of the results.\n\nFor example, the study of Morsc et al (26) is wrongly cited and interpreted. The authors state \"Morsch et al. concluded that pyridostigmine remains the only approved symptomatic drug for the treatment of BoNT intoxication, and one week of ingestion was enough to exacerbate neuromuscular impairment (26)“. This statement cannot be found in the cited publication.\n\nAnother miscitation refers to reference 27. The authors state “Similarly, there were significant differences in the weights of the tibialis muscle between groups in different study periods“, while in the cited study (27) five times higher dose of pyridostigmine (25 mg/kg) was applied via infusion pump and did not measure tibialis muscle weight.\n\nHow does the last paragraph in the Discussion section relate to the aim and the results of the present study?\nConclusion:\nThe conclusion must include novelty - how the study contributes to what is known in the field, study limitations, and the potential clinical significance of the results.\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Partly\n\nAre sufficient details of methods and analysis provided to allow replication by others? Partly\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nI cannot comment. A qualified statistician is required.\n\nAre all the source data underlying the results available to ensure full reproducibility? Partly\n\nAre the conclusions drawn adequately supported by the results? No",
"responses": []
}
] | 1
|
https://f1000research.com/articles/11-1547
|
https://f1000research.com/articles/11-1545/v1
|
21 Dec 22
|
{
"type": "Case Report",
"title": "Case Report: Endotracheal tube as life-saving tool to control an aortic rupture in intensive care unit",
"authors": [
"Ciro Campanella",
"Mohannad Abbass",
"Umberto Marzi",
"Franco Masini",
"Salvatore Lentini",
"Mohannad Abbass",
"Umberto Marzi",
"Franco Masini",
"Salvatore Lentini"
],
"abstract": "Background: Aortic rupture and suture dehiscence can complicate a cardiac operation, especially in case of infections of the surgical site. Such a complication can be life-threatening and require immediate surgical treatment. Case: We report the case of a 13-year-old girl who suffered a sudden dehiscence of the aortic suture line in the context of deep sternal wound infection, while in the surgical intensive care unit after a double valve replacement. Control of bleeding was obtained by the insertion of an endotracheal tube into the ascending aorta and inflation of the tube cuff to plug the aortic bleeding point. The extracorporeal circulation was then established and under deep hypothermic arrest the defect was successfully repaired. The patient was discharged 14 days after surgery and reviewed at the outpatient clinic in good health. Conclusions: An endotracheal tube can be used in cases of uncontrollable aortic bleeding as a life-saving tool to bridge the patient to adequate surgical treatment.",
"keywords": [
"Aortic rupture",
"deep sternal wound infection",
"cardiac surgery",
"haemorrhage"
],
"content": "Introduction\n\nSudden aortic rupture after cardiac surgery is a life-threatening complication that can lead to death.1 Aortic rupture or dehiscence of an aortic suture can happen also as a consequence of post-cardiotomy deep sternal wound infections (DSWI), but reported outcomes of these cases are poor.2 Diagnosis and treatment of such a complication can be challenging, especially if the sudden rupture happens outside the operating theatre where the establishment of an emergent surgical procedure is difficult. It is, thus, necessary to identify simple life-saving tools to control the aortic bleeding while bringing the patient to surgery. Herein, we report the case of a post-cardiotomy DSWI complicated by sudden dehiscence of the aortic suture controlled with the use of an endotracheal tube.\n\n\nCase report\n\nThis case report follows the CARE guidelines [8]. A 13-year-old girl from North Sudan was referred to our hospital for palpitations, fatigue, lower limb oedema and shortness of breath. Echocardiography showed severe aortic and mitral valve regurgitation with mildly reduced left ventricular function in the context of rheumatic heart disease. The patient underwent aortic and mitral valve replacement using a 19 mm SJM Regent mechanical prothesis (Abbott, Burlington, MA USA) in aortic position and a 27 mm SJM Regent mechanical prothesis (Abbott, Burlington, MA USA) in mitral position. The procedure was unremarkable, and the aorta was closed with a double layer technique.\n\nThe post-operative course was characterized by high fever (39.2°C) and inflammatory markers (white blood cells count: 15.75 10^3/uL; C-reactive protein: 248 mg/L) during the first post-operative day (POD). The patient was extubated on POD 4 but continued to be febrile with blood and wound cultures positive for Klebsiella pneumoniae. Chest x-rays showed signs of pneumonia. She was treated with empirical antibiotic therapy (amikacin 450 mg/150 ml once a day; ceftazidime 1.5 mg/50 ml three times a day) for 2 days and switched to piperacillin/tazobactam (4.5 g/50 ml continuous infusion at 4 ml/h) based on the antibiotic sensitivity. On POD 8 the sternal wound appeared swollen and oedematous with minimal discharge and 2 days later the patient was taken back to the operating theatre for wound re-exploration. The sternal wound was reopened and the diagnosis of DSWI was confirmed. Careful debridement was carried out. The sternum and skin were left open, swabs soaked in diluted chlorhexidine were left in place.\n\nThe wound rapidly improved in the subsequent 48 hours. On the day before the planned surgical closure, the patient developed a sudden bleeding through the drains. The swabs covering the heart were urgently removed in the intensive care unit (ICU) and active bleeding was noted from the aortotomy suture line. Digital pressure secured partial control, but the patient developed haemorrhagic shock.\n\n\nSurgical technique\n\nAn attempt of placing a partial occluding clamp on the ascending aorta failed due to the rigid and inflamed aortic wall. Furthermore, a partial occluding clamp could have compromised the systemic and coronary blood flow. As an alternative strategy, a number 6 endotracheal tube (ENT Medical Instrument Co. LTD, China) was inserted into the aortic lumen through the aortic dehiscent suture line and its cuff was inflated with 10 ml of saline (Figure 1A). By pulling the tube through the aortic opening, the inflated cuff accommodated itself inside the perimeter of the aortic opening securing a haemostatic seal and preventing occlusion of the aortic lumen and coronary ostia. It was then possible to see that the aortic suture line had cut through the aortic wall, generating a gap of around 2x2 cm.\n\nRepresentation of the main steps of the procedure to use an endotracheal tube as life-saving tool to control an aortic rupture in intensive care unit: temporary occlusion of the aortic tear with the use of an endotracheal tube (A) and subsequent closure of the tear with patch (B).\n\nThe left femoral artery and the right atrium were cannulated, and cardiopulmonary bypass (CPB) was established. The patient was cooled to 24°C and deep hypothermic circulatory arrest (DHCA) with exsanguination (flow 500 ml/min) was established for 16 minutes (total CPB time 148 minutes). This allowed the avoidance of a crossclamp and cardioplegia administration, reducing the risks for further aortic wall damage. While on DHCA, the endotracheal cuff was deflated, and the tube removed. The patient was positioned in the Trendelenburg position and a pump sucker was inserted through the opening in the aortic wall and advanced toward the aortic arch. A pericardial patch of 2x1.5 cm was secured along the perimeter of the aortic dehiscence with a continuous 5/0 Prolene (Figure 1B). The suture line was reinforced using surgical glue. Finally, the patient was weaned off CPB. Of note, the whole surgery was performed at the ICU bed.\n\nThe patient was left with the chest open for a further 48 hours. She remained stable through that period and underwent delayed chest closure. She was weaned from mechanical ventilation 72 hours after the last surgery and was discharged home 2 weeks later. She was reviewed at the outpatient clinic 4 weeks after the original procedure in good health.\n\n\nDiscussion\n\nSudden aortic rupture is a catastrophic event that generally ends in patient death.1,2 This case report illustrates not only the complications of a DSWI, but also the possibility to improvise major cardiac surgical procedure outside the operating theatre. To the best of our knowledge, this is the first report depicting the use of an endotracheal tube as haemostatic plug to control major bleeding from large vessels and one of the few reports of aortic rupture as consequence of DSWI.\n\nDSWI is a severe complication after cardiac surgery, with a reported incidence ranging from 0.2 to 8.0%3,4 and early mortality rates from 7.3% to 21.6%.5,6 Patients with DSWI have significantly higher overall mortality, in-hospital mortality, follow-up mortality, and major adverse cardiovascular events compared with patients without DSWI.7 While risk analyses and outcomes of DSWI have been widely investigated, the literature lacks detailed studies on causes of mortality. Moreover, very few reports are available regarding the consequences of a DSWI on aortic walls, especially after aortic surgery. In our case, the major bleeding occurred from the aortotomy site. The aggressive DSWI probably exacerbated an aortitis, which led to thinning and perforation of the weakest point of the aortic wall as previously described by Kim et al.2\n\nSudden aortic rupture has a fatal outcome in most cases.1,2 In our case a combination of “fortuitous” events allowed for a successful outcome. Firstly, the patient’s chest was already open and when the aortic suture line started bleeding it was sufficient to remove the swabs to confirm the diagnosis and apply a digital pressure. With a closed chest, time-lapse between the visualisation of the blood in the drainage bottles and the diagnosis would have resulted in the patient’s exsanguination. Furthermore, the decision to keep the patient at the ICU bed and call instead for the theatre staff and perfusionist personnel to help, proved extremely efficient allowing for emergency cannulation and CPB establishment in very few minutes. Finally, we feel that the unconventional use of an endotracheal tube proved vital to stop the massive bleeding and gave us the time to proceed to establish the CPB. Nevertheless, this is an anecdotical report on an unconventional technique to control aortic bleedings. As such, it is limited by a difficult reproducibility and by the off-label use of the endotracheal tube. Thus, this technique should be considered only as bail-out approach in case of lack of other conventional techniques.\n\nIn conclusion, this case illustrates that the management of a major uncommon complication in cardiac surgery is possible even in challenging logistical circumstances. However, further studies are required to clarify this.\n\n\nConsent\n\nWritten informed consent for publication of their clinical details was obtained from the father 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\nFigshare: CARE checklist for ‘Case Report: Endotracheal tube as life-saving tool to control an aortic rupture in intensive care unit’. https://doi.org/10.6084/m9.figshare.21617565. 8\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\nKimura N, Tanaka M, Kawahito K, et al.: Early postoperative aortic rupture following surgery for acute type A aortic dissection. Interact. Cardiovasc. Thorac. Surg. 2009; 8(4): 431–434. Publisher Full Text\n\nKim SW, Lee S, Chang JW: Delayed aortic rupture resulting from postoperative superficial sternal wound infection. J. Thorac. Dis. 2016; 8(7): E523–E526. PubMed Abstract | Publisher Full Text | Free Full Text\n\nSchimmer C, Reents W, Berneder S, et al.: Prevention of sternal dehiscence and infection in high-risk patients: a prospective randomized multicenter trial. Ann. Thorac. Surg. 2008; 86(6): 1897–1904. PubMed Abstract | Publisher Full Text\n\nLo Torto F, Turriziani G, Donato C, et al.: Deep sternal wound infection following cardiac surgery: A comparison of the monolateral with the bilateral pectoralis major flaps. Int. Wound J. 2020; 17(3): 683–691. PubMed Abstract | Publisher Full Text | Free Full Text\n\nPerrault LP, Kirkwood KA, Chang HL, et al.: A Prospective Multi-Institutional Cohort Study of Mediastinal Infections After Cardiac Operations. Ann. Thorac. Surg. 2018; 105(2): 461–468. PubMed Abstract | Publisher Full Text | Free Full Text\n\nGummert JF, Barten MJ, Hans C, et al.: Mediastinitis and cardiac surgery--an updated risk factor analysis in 10,373 consecutive adult patients. Thorac. Cardiovasc. Surg. 2002; 50(2): 87–91. PubMed Abstract | Publisher Full Text\n\nPerezgrovas-Olaria R, Audisio K, Cancelli G, et al.: Deep Sternal Wound Infection and Mortality in Cardiac Surgery: A Meta-analysis. Ann. Thorac. Surg. 2022. PubMed Abstract | Publisher Full Text\n\nLentini S: Case Report: Endotracheal Tube as Life-saving Tool to Control an Aortic Rupture in Intensive Care Unit. [Dataset]. figshare 2022. Publisher Full Text"
}
|
[
{
"id": "158587",
"date": "17 Jan 2023",
"name": "Justine Mafalda Ravaux",
"expertise": [
"Reviewer Expertise Pacemaker implantation",
"rhythm disturbances",
"heart mechanical support",
"ECMO",
"coronary artery disease",
"aortic disease",
"sternal wound infection",
"mitral regurgitation"
],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nCampanella and colleagues presented a single case report experience of an endotracheal tube used as a plug for managing an aortic rupture outside in an intensive care unit. The progression of the issue’s management is clearly described and the authors provided sufficient background in the field of interests. Moreover, details about diagnostic test, procedure (surgical technique, logistic…) are correctly mentioned in the manuscript. The discussion emphasizes the feasibility of the technique when performing high-risk interventions in a “hostile” environment, allowing other practitioners to consider such an option when dealing with a similar problem. The manuscript is overall well-written and no English revision is needed. If we think of the current intra-aortic balloon used for mini-invasive surgical procedures (Intraclude, Edwards Lifesciences Inc), this case report sounds a little bit like a “Back to Basics”, which may be helpful in a non-surgical environment. The authors should be congratulated for reporting such a kind of “home-made” solution, when the scientific community is globally focusing on the development of new complex devices and/or techniques, while the focus should be on the implementation of all these techniques worldwide, avoiding two-speed medicine. Herewith enclosed are my minor comments.\nAbstract, line 7. “(…)while in the surgical intensive care(…)”. Please rephrase with “(…)occurring at the intensive care(…)”.\n\nAbstract, lines 15-17. I would emphasize that these techniques may especially be considered in a non-surgical environment.\n\nIntroduction, line 7. I would add to the last sentence that this manoeuvre was performed at the intensive care unit.\n\nCase report, line 9. The authors mentioned positive wound culture for Klebsiella Pneumonia. Did the patient experience a leak from the sternal wound with pus or other sign of wound infection, like dehiscence of the wound? Please specify.\n\nSurgical technique. After placing the endotracheal tube to control the aortic bleeding at the intensive care unit, why did the team not transfer the patient into the operating room for further surgical management? Was the patient too hemodynamically unstable or was it easier (in the way, faster) to ask the whole team to come to the intensive care unit?\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": "296771",
"date": "08 Nov 2024",
"name": "Jigisha Pujara",
"expertise": [
"Reviewer Expertise Cardiac Anesthesia related articles and critical care"
],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis case report is an interesting case, in which authors have described the life saving technique for post Double valve replacement complication of sternal wound infection followed by aortic dehisence. Authors have described the innovative technique to control the bleeding by endotracheal tube cuff, to save the life and work as a bridge to definitive repair of aortic rupture. this case report can help other cardiac surgeons to do the similar management if they face same complication. Only suggestion to author is, kindly recheck the antbiotic dosages,which are not correct or typographical error> please recheck them and correct. (ideally dosage to be written as per kg dose).\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/11-1545
|
https://f1000research.com/articles/11-1544/v1
|
21 Dec 22
|
{
"type": "Genome Note",
"title": "Draft genome of a clinical Pseudomonas qingdaonensis isolate from a hospitalized patient at Kenyatta National Hospital, Kenya",
"authors": [
"Brian Ogoti",
"Racheal Gachogo",
"Frank Onyambu",
"Racheal Gachogo",
"Frank Onyambu"
],
"abstract": "The first draft genome of Pseudomonas qingdaonensis, a gram-negative bacteria isolated from hospitalized patients in Kenya, is presented in this study. The genome was assembled using nanopore MinION readings, yielding an assembly of 8,857,650 base pairs made up of 5(five) contigs. The genome sequence of Pseudomonas qingdaonensis will help researchers better grasp its genetics. Furthermore, the data can be a valuable resource for comparative genomics and future research in this novel species.",
"keywords": [
"Draft genome",
"Pseudomonas qingdaonensis"
],
"content": "Abbreviations\n\nMLST: Multilocus sequence typing\n\nrRNA: Ribosomal RNA\n\ntRNA: Transfer RNA\n\n\nIntroduction\n\nThe diverse Gram-negative genus Pseudomonas contains species isolated from varied habitats, plants, animals, and people. It is a gram negative, aerobic, rod-shaped belonging to the gammaproteobacteria.1 Here we describe a novel species Pseudomonas qingdaonensis isolated from a hospitalized patient in Kenya. Its genome sequencing will aid in our understanding of this pathogen's biology. The draft genome assembled here will enable more in-depth studies to be conducted in specific genome sections or genes that promote the novel Pseudomonas species reported here in the colonization of hospitalized patients.\n\n\nMethods\n\nThe Pseudomonas qingdaonensis was isolated from a hospitalized patient at Kenyatta National hospital in Kenya. A stool sample was collected from the hospitalized patient and in the laboratory, it was cultured on MacConkey agar (HiMedia Lab, Mumbai, India) at 37°C for 18 hours. The colonial morphology on MacConkey agar plate, catalase, oxidase and gram staining procedure tests were used in identification.2 The colonies on MacConkey were flat, 2-3mm, smooth colonies with an irregular leafy margin. Under the microscope after gram staining procedure, gram negative rods measuring 0.6-0.8 μm were identified. On the biochemical tests, the colonies isolated were catalase positive, and oxidase positive. Confirmation of the ID of Pseudomonas species was done using the VITEK® 2 (Biomerieux, France). Before loading isolates into the VITEK® 2, bacterial suspensions were done by emulsifying the isolates in 0.5% saline and standardizing turbidity to 0.5 McFarland’s using a densitometer. The suspension was used for species ID and AST in the VITEK® 2 using Gram-negative ID cards (ID-GN 21341) and results analyzed according to Clinical and Laboratory Standards Institute guidelines.3\n\nA single colony was sub-cultured on nutrient agar (HiMedia Lab, Mumbai, India) at 37°C for 18 hours. Colonies from nutrient agar were resuspended in 400 ul of phosphate buffered saline and then extracted using ISOLATE II Genomic DNA Kit (Meridian Bioscience Inc, London, UK). The DNA was eluted with pre-warmed nuclease free water in a total volume of 50 ul. Sequencing library preparation was done using the Oxford Nanopore genomic sequencing kit SQK-LSK109 (ONT, UK) and Native Barcoding expansion kits (EXP-NBD104, EXP-NBD114) as per the manufacturer’s instructions. Sequencing was performed using the MinKNOW software (ONT, United Kingdom, Oxford) with live Base calling turned on. The quality score of the FAST5 and subsequent FASTQ files produced by MinKNOW was set at 7.\n\nLive base calling and adapter trimming was performed using Guppy v3.6.1 (https://nanoporetech.com). De-novo assembly of the Pseudomonas qingdaonensis was performed using UNICYCLER v0.4.9 (https://github.com/rrwick/Unicycler).4 The genome quality and assembly was contrasted and carefully selected based on the following criteria: the size of contigs, GC (%) content, N50 length, and genome coverage as determined by QUAST v5.2.0 (http://bioinf.spbau.ru/quast). Visualization of the draft assembly was done using Bandage v0.9.0 (https://rrwick.github.io/Bandage).5 Genome annotation of the draft assembly was done using the NCBI Prokaryotic Genome annotation pipeline (PGAP) (https://www.ncbi.nlm.nih.gov/genome/annotation_prok/).\n\nThe Kenyatta National Hospital – University of Nairobi (KNH-UON) Ethics and Research Committee was sought for approval and approved under Ref no. P391/07/2020. Signed informed consent, approved under Ref no. P391/07/2020, was obtained from the participant and the project followed ethical principles and guidelines for research involving human subjects.\n\n\nResults\n\nThe total length of the Pseudomonas qingdaonensis was 8,857,650 base pairs long consisting of 5(five) contigs. The N50 contig was 3332773 and the GC% content was 64.03% (Table 1). The total coverage of the draft genome was 51X.\n\nThe draft genome was submitted to NCBI GenBank repository under the accession number JANWGM000000000.1. The genome annotation done using PGAP showed the presence 6,274 genes with 3,090 being coding genes, 19 rRNAs and 70 tRNAs (Table 2).\n\nThe draft sequence of Pseudomonas qingdaonensis was submitted to the PubMLST website for multilocus sequence typing (https://pubmlst.org/) to generate an in-silico MLST profile. Table 3 show the allelic matches from PUBMLST.\n\n\nLimitations\n\nComparative analyses were not undertaken, and more research is needed to identify Pseudomonas qingdaonensis relationship to other Pseudomonas qingdaonensis isolates globally.",
"appendix": "Data availability\n\nGenBank: Pseudomonas qingdaonensis, whole genome sequence, Accession number JANWGM000000000.1: https://www.ncbi.nlm.nih.gov/Traces/wgs/JANWGM01?display=contigs\n\nBioProject: Pseudomonas qingdaonensis strain: CMB_001, Accession number PRJNA869907: https://identifiers.org/NCBI/bioproject:PRJNA869907\n\nBioSample: Pathogen: clinical or host-associated sample from Pseudomonas qingdaonensis, Accession number SAMN30333618: https://identifiers.org/biosample:SAMN30333618\n\n\nAcknowledgements\n\nWe acknowledge the Centre of Molecular Biosciences for providing the resources to sequence the genome.\n\n\nReferences\n\nWang MQ, Wang Z, Yu LN, et al.: Pseudomonas qingdaonensis sp. nov., an aflatoxin-degrading bacterium, isolated from peanut rhizospheric soil. Arch. Microbiol. 2019 Jul; 201(5): 673–678. PubMed Abstract | Publisher Full Text\n\nGiuliano C, Patel CR, Kale-Pradhan PB: A Guide to Bacterial Culture Identification And Results Interpretation. Pharm. Ther. 2019 Apr; 44(4): 192–200.\n\nClinical and Laboratory Standards Institute: CLSI: Performance Standards for Antimicrobial Susceptibility Testing. Twenty-Seventh Informational Supplement. CLSI Document M100-S27. [Internet]. 2017; p. 282.Reference Source\n\nCompleting bacterial genome assemblies with multiplex MinION sequencing. Microbiology Society. [cited 2022 Dec 7]. Publisher Full Text\n\nWick RR, Schultz MB, Zobel J, et al.: Bandage: Interactive visualization of de novo genome assemblies. Bioinformatics. 2015; 31(20): 3350–3352. PubMed Abstract | Publisher Full Text | Free Full Text"
}
|
[
{
"id": "158492",
"date": "03 Jan 2023",
"name": "Gerald Mboowa",
"expertise": [
"Reviewer Expertise Bioinformatics & Genomics"
],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nTake note that this is written as Gram-negative not gram-negative throughout the entire write up.\nAuthors should include the following to make this work more informative:\nWhere was sequencing done? Annotation of antimicrobial resistance genes and virulence factors should be included Clinical history and demographics of the patient\nInclude a brief discussion of the findings from this genome assembly.\n\nAre the rationale for sequencing the genome and the species significance clearly described? Yes\n\nAre the protocols appropriate and is the work technically sound? Yes\n\nAre sufficient details of the sequencing and extraction, software used, and materials provided to allow replication by others? Yes\n\nAre the datasets clearly presented in a usable and accessible format, and the assembly and annotation available in an appropriate subject-specific repository? Yes",
"responses": []
},
{
"id": "168571",
"date": "02 May 2023",
"name": "Aquillah Kanzi",
"expertise": [
"Reviewer Expertise Genomics",
"structural bioinformatics",
"genetics and microbiology"
],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe authors sequenced and assembled a draft genome of Pseudomonas qingdaonensis isolated from a hospitalised patient. The methodology used for sequencing and assembly is technically sound. The comments and suggestions below can improve the quality of this work.\nIntroduction 1. The authors use of \"novel species\" in the introduction is misleading.\nBio-informatics analyses 2. The authors used QUAST to assess the quality of the assembled genome. Was a reference genome used? e.g. GCF_021601925.1\nResults 3. The stated size of the draft genome in the results section 8,857,650 differs from the QUAST statistics in table 1.\n4. There is no visual presentation of the draft genome. Visualisation was described in the Bio-informatics analyses section but no genome map was shown. A visual presentation will improve the quality of this work\n5. The results shown in Table 3 are not comprehensible. Yes, there are matching MLST profiles in PUBMLST but what does this mean?\nThe article needs a short discussion section to put into context the main findings.\n\nAre the rationale for sequencing the genome and the species significance clearly described? Yes\n\nAre the protocols appropriate and is the work technically sound? Yes\n\nAre sufficient details of the sequencing and extraction, software used, and materials provided to allow replication by others? Yes\n\nAre the datasets clearly presented in a usable and accessible format, and the assembly and annotation available in an appropriate subject-specific repository? Yes",
"responses": []
}
] | 1
|
https://f1000research.com/articles/11-1544
|
https://f1000research.com/articles/11-1543/v1
|
21 Dec 22
|
{
"type": "Research Article",
"title": "Diurnal small RNA expression and post-transcriptional regulation in young and old Drosophila melanogaster heads",
"authors": [
"Rosalyn M. Fey",
"Eileen S. Chow",
"Barbara O. Gvakharia",
"Jadwiga M. Giebultowicz",
"David A. Hendrix",
"Eileen S. Chow",
"Barbara O. Gvakharia",
"Jadwiga M. Giebultowicz"
],
"abstract": "Background: MicroRNAs are a class of small (~22nt) endogenous RNAs that regulate target transcript expression post-transcriptionally. Previous studies characterized age-related changes in diurnal transcript expression but it is not understood how these changes are regulated, and whether they may be attributed in part to changes in microRNA expression or activity with age. Diurnal small RNA expression changes with age were not previously studied. Methods: To interrogate changes in small RNA expression with age, we collected young (5 day) and old (55 day) Drosophila melanogaster around-the-clock and performed deep sequencing on size-selected RNA from whole heads. Results: We found several microRNAs with changes in rhythmicity after aging, and we investigated microRNAs which are differentially expressed with age. We found that predicted targets of differentially expressed microRNAs have RNA-binding and transcription factor activity. We used a previously published method to identify mRNA transcripts which show evidence of microRNA targeting that is altered after aging, and found several that are involved in muscle development and maintenance. Finally, we identified novel microRNAs using the random-forest-based method miRWoods, which surprisingly also discovered transfer RNA-derived fragments. Conclusions: We showed a decrease in global microRNA expression and a corresponding increase in piRNA expression during aging. We also found an increase in rhythmicity of Drosophila small RNAs during aging, including microRNAs, piRNA clusters, and novel transfer RNA-derived fragments. To our knowledge this is the first study examining diurnal small RNA expression around the clock in young and old Drosophila, and as such it paves the way for future research on changes in small RNA regulatory molecules in the context of aging.",
"keywords": [
"microRNA",
"transfer RNA-derived fragments",
"piRNA",
"circadian",
"diurnal",
"small RNA",
"aging"
],
"content": "Introduction\n\nDiurnal transcript expression is controlled by daily light-dark cycles or by the circadian clock, a conserved molecular feedback loop that regulates daily physiological and behavioral rhythms and is altered during normal aging (Popa-Wagner et al. 2017; Rakshit et al. 2012). Changes in the expression of the diurnal transcriptome with age, including loss and gain of 24-hour rhythmicity, have been shown in multiple organisms, including fruit flies (Kuintzle et al. 2017), zebrafish (Park and Belden 2018), and humans (Chen et al. 2016). While age-related changes in gene expression may impact health and longevity (Acosta-Rodriguez et al. 2021), the causes of shifts in the diurnal transcriptome are not understood. In young flies, transcript rhythmicity is regulated in part by post-transcriptional mechanisms, including regulation by microRNAs (Xue and Zhang 2018). MicroRNAs (miRNAs, miRs) are a class of small (~22 nucleotide) regulatory RNA molecules that cause degradation or translational repression of target mRNAs, altering their level of expression in the cell (Bartel 2018). However, it is not known whether diurnal microRNA profiles change with age and, if so, whether such changes may be involved in altering diurnal transcriptome profiles in old organisms. Here, we address these questions in the fruit fly Drosophila melanogaster.\n\nMetazoan microRNAs are transcribed as primary transcripts (pri-microRNA) that fold into a hairpin structure, are cleaved into a pre-microRNA by the nuclear enzyme Drosha, exported into the cytoplasm, and processed into a microRNA duplex by the enzyme Dicer (Figure 1A) (Bartel 2018). The two strands of the duplex are separated into 5' and 3' arms, one of which is a generally more highly-expressed functional microRNA (miR), and one of which is a largely non-functional product known as the “miR star” (miR*) (Bartel 2018). The functional miR is bound by the protein Ago1 and incorporated into the RNA-induced silencing complex (RISC), while the miR* is degraded in the cell (Bartel 2018). The RISC binds a sequence in the 3' untranslated region (UTR) of the target mRNA with partial base pair complementarity to the seed region (nucleotides 2–8) of the microRNA, causing degradation or translational repression of the mRNA (Bartel 2018).\n\nMicroRNAs are well-studied in Drosophila, and several experiments have provided evidence for the role of microRNAs in circadian and light-activated regulation of gene expression in young flies. Yang et al. demonstrated circadian regulation of miR-263a and miR-263b (Yang et al. 2008), while miR-124 has been shown to modulate phase locomotor activity, a circadian clock output behavior (Zhang et al. 2016). Other microRNAs target core circadian clock genes Clk (Kadener et al. 2009) and cwo (Chen et al. 2014) (bantam and let-7, respectively). Further, miR-276a, which targets the core clock gene tim, was shown to be indirectly light-activated (Chen and Rosbash 2016). A miR-959-964 cluster, which shows strong diurnal oscillations, regulates the phase of feeding and immune function (Vodala et al. 2012).\n\nAlthough microRNAs are known regulators of development (Chandra et al. 2017), they also play important roles in aging and neurodegeneration. For example, miR-34 expression increases in the Drosophila brain during normal aging, and it is involved in the regulation of neurodegeneration and lifespan (Liu et al. 2012). The co-transcribed microRNAs miR-125, let-7 and miR-100 also increase in expression with age, with miR-125 and let-7 required for normal lifespan (Chawla et al. 2016). Many other studies have revealed roles for individual microRNAs in aging and neuroprotection (reviewed in (Kinser and Pincus 2020)).\n\nAnother class of Drosophila small RNA that may have a role in aging and neurodegeneration is Piwi-interacting RNAs (piRNAs), 23 to 29 nucleotide molecules that target transposons in the germline (Brennecke et al. 2007) and also function in somatic tissues, including the brain (Wakisaka and Imai 2019; Kim 2019). Transposons are mobile genetic elements which promote genetic diversity; however, if left unchecked to insert in the genome, will result in hybrid dysgenesis and mutations in flies (reviewed in (McCullers and Steiniger 2017)). The biogenesis of piRNAs falls into two categories: the production of primary piRNAs, whereby a piRNA precursor is transcribed from the genome and processed into mature piRNAs, and the production of secondary piRNAs using a primary piRNA as a template (Figure 1B) (Czech and Hannon 2016). Secondary piRNAs act post-transcriptionally to silence active transposons (Czech and Hannon 2016).\n\nTransposon expression and activity increase during aging in flies (Li et al. 2013), and as piRNAs have been shown to target transposons to reduce their activity, it is hypothesized that piRNAs play a protective role against transposon-induced genome damage during aging. While studies investigating piRNAs in the circadian system are sparse, previous work in our lab has shown that five putative piRNA-containing transcripts show both increased expression level and increased diurnal rhythmicity in the heads of aged flies (Kuintzle et al. 2017).\n\nAlthough many Drosophila small RNAs are well-studied as individual regulators in young animals, their systems-level role in circadian or light-activated regulation of transcript expression during aging is not understood. To assess the relationship between diurnal small RNA expression and the aging circadian clock output, we performed small RNA sequencing around-the-clock on the heads of young (5 day) and old (55 day) female D. melanogaster. In this work we analyzed age-related expression and rhythmicity changes in microRNAs and piRNAs, and investigated the potential biological effects of microRNA expression changes with age by integrating this small RNA dataset with an RNA-seq dataset previously published by our lab (Kuintzle et al. 2017). In addition, we identified novel age-onset microRNAs and transfer-RNA derived fragments (tRFs) and characterized changes in their expression during aging.\n\n\nMethods\n\nThe animals used in this study do not require ethics approval in accordance with Oregon State University’s Institutional Animal Care and Use Committee.\n\nMated female Drosophila melanogaster (w1118) were mainatined at 25°C on a standard agar (7.4g/L) (Genesee Scientific, Catalog number: 66-103), cornmeal (50g/L) (Genesee Scientific, Catalog number: 62-101), yeast (35g/L) (Genesee Scientific, Catalog number: 62-103), and molasses (5%) (Genesee Scientific, Catalog number: 62-117) diet under a light-dark (LD) 12h:12h cycle and housed in groups of 50 flies in 300 ml round bottom polypropylene ventilated bottles (Genesee Scientific, Catalog number: 32-129F). Diet was changed without anesthesia three times a week. Two biological replicates of 400 flies were collected for young and old at 4 hour intervals around the clock. In parallel with samples for sequencing, 50 flies young and old were collected at each time point for potential verification of data by qPCR. At each time point, flies were transferred without anesthesia to Eppendorf tubes (Genesee Scientific, Catalog number: 22-282) that were pre-frozen at -80°C. Whole flies were stored at -80°C. Heads were separated from bodies by vortexing tubes (Scientific Industries Vortex-Genie 2, Catalog number: SI-0236) and sifting them over stainless steel sieves with mesh opening sizes of 710 μm and 425 μm (custom sieves ordered from Tokyo Screen Co). Samples were kept frozen throughout the process with liquid nitrogen, and stored at -80°C until RNA isolation was performed.\n\nEach sample of 400 heads was homogenized in TRIzol (Thermo Fisher, Catalog number: 15596026) with a handheld motorized pestle (motor: Kimble, Catalog number: 749540-0000; pestle: Kontes, Catalog number: KT-749521-1590) following the manufacturer’s instructions. Samples were treated with rDNAse I (Takara, Catalog number: 2270A) following the manufacturer’s instructions, and then extracted with a 1:1 ratio of sample volume tophenol/chloroform (Thermo Fisher, Catalog number: 15593031) volume. RNA was then precipitated with 200 proof ethanol (using 2.5x of RNA sample volume) (Koptec, Catalog number: 2716) and 0.3M pH 5.5 sodium acetate (using 0.1x of RNA sample volume) (Invitrogen, Catalog number: AM9740). Finally, the resulting RNA pellet was rinsed twice with 75% ethanol (Koptec, Catalog number: 2716) and dissolved in 50 μl nuclease-free water (Invitrogen, Catalog number: AM9939). The concentration and purity of total RNA was assessed with a NanoDrop NanoDrop® ND-1000 (Thermo Fisher). RNA was extracted in the same way from 50 heads per sample, except the final RNA pellet was dissolved in 20 μl nuclease-free water instead of 50 μl, and allocated for qPCR.\n\nDrosophila small RNA sequencing experiments are known to be overwhelmed by a 30-nucleotide rRNA fragment, if the fragment is not depleted (Wickersheim and Blumenstiel 2013). Therefore, we size-separated total RNA from each sample using 15% denaturing polyacrylamide-urea gel (polyacrylamide: Bio-Rad, Catalog #1610144; urea: Thermo Fisher, Catalog # 15505035). The gel was pre-run in 0.5X TBE buffer at a constant current of 40 mA for 30 minutes, and then samples were loaded and run at a constant current of 30 mA for approximately 1 hour. The gel was stained for 15 minutes with SYBR™ Gold Nucleic Acid Gel Stain (Thermo Fisher; Catalog number: S11494) in 0.5X TBE buffer following the manufacturer’s instructions, then small RNA in the size range of 18 to 29 nucleotides were cut out and gel fragments were mixed with 1 μl of glycogen (Thermo Fisher, Catalog #9510) as a carrier. RNA was eluted overnight using 0.3M sodium acetate precipitation (Invitrogen, Catalog number: AM9740). RNA was then precipitated with 200 proof ethanol (using 3x of the sample volume) (Koptec, Catalog number: 2716). The resulting RNA pellet was rinsed twice with 75% ethanol (Koptec, Catalog number: 2716) and dissolved in 7 μl nuclease-free water (Invitrogen, Catalog number: AM9939). Full protocol details are available on protocols.io (DOI: https://dx.doi.org/10.17504/protocols.io.yxmvm2xpog3p/v1) (Fey et al. 2022).\n\nPrior to library preparation, the quality and concentration of the size-selected small RNA was assessed with a Qubit 3.0 fluorometer (Thermo Fisher, Catalog number: Q33216) and the appropriate Qubit microRNA assay kit (Thermo Fisher, Catalog number: Q32880) according to the manufacturer’s instructions. To ensure complete depletion of the 30-nucleotide rRNA fragment, a DNA oligo with sequence complementarity to the fragment (Wickersheim and Blumenstiel 2013) was added to samples during the library preparation stage (1–2 μl of 10 μM oligo stock was added to each sample of 10–50 ng RNA in 5μl).\n\nLibraries were prepared using TruSeq Small RNA Library Prep Kit (Illumina; Catalog number: RS-200-0012) following the manufacturer’s instructions. Briefly, adapters were ligated to the 3' and 5' ends of the sample and reverse transcription followed by amplification created cDNA constructs based on the small RNA ligated with 3' and 5' adapters. This step selectively enriches RNA fragments with adapter molecules on both ends. The amplified cDNA constructs were pooled, then gel purified. The final pooled library was quantified by Qubit fluorometer using the dsDNA HS Assay kit (Thermo Fisher; Catalog number: Q33230). For quality control analysis, 1 μl of resuspended construct was loaded on an Agilent Technologies 2100 Bioanalyzer using a DNA-specific chip. Library pools were quantified by qPCR using an ABI 7500 fast instrument and KAPA Biosystems Library Quantification kit (KAPA Biosystems; Catalog numer: KK4824). Libraries were run on an Illumina HiSeq 3000 instrument, 50bp single end with 12 libraries per lane.\n\nReads were quality filtered and sequencing adapters were trimmed using skewer (Jiang et al. 2014), with parameters set for a minimum quality score of 30 and a minimum read length of 18, using the adapter sequence “TGGAATTCTCGGGTGCCAAGG”. Reads were aligned to the Drosophila melanogaster genome (BDGP release 6.21/dm6) with Bowtie using the parameters “-l 18 –n 1 -e 50 -a -m 50 -S --best --strata”. The number of hits to the genome for each read was included by adding NH tags to the output SAM files.\n\nWe used a custom Python script to quantify reads by mapping to the miRBase version 22 genome annotation file (release 6) (Kozomara, Birgaoanu, and Griffiths-Jones 2019). Of the 495 microRNAs annotated in mirBase, we detected 458 in our experiment. We adjusted reads to account for paralogous microRNAs (which map to multiple places in the genome) by dividing each count by the number of hits to the genome, stored in the NH tags in the SAM file for each sample. Final quantification is in adjusted Reads per Million Mapped MicroRNAs (aRPMMM). We used similar custom Python scripts to quantify reads mapping to piRNAs.\n\nDiurnal rhythmicity was computed using a Fourier-based method previously developed by our lab, whereby the relative power of the 24-hour period (RP24) is used as a measure of diurnal rhythmicity (Sebastian et al. 2022). We calculated an RP24 score for each microRNA using their around-the-clock expression profiles and calculated statistical significance for microRNAs with RP24 scores ≥ 0. MicroRNAs with q-values ≤ 0.05 were considered significantly rhythmic, and microRNAs with q-values > 0.05 were considered arrhythmic. Rhythmic transcripts were identified previously using the RP24 method (Sebastian et al. 2022).\n\nWe compared the global expression of small RNAs in young and old flies using a t-test, implemented with SciPy tools version 1.0.0 (Jones et al. 2001). All 12 samples in young flies comprised one group, and all 12 samples in old flies comprised another group. The means of the two groups were considered significantly different with a p-value ≤ 0.05.\n\nWe performed differential expression analysis using the programs edgeR (Robinson and Oshlack 2010) and DESeq2 (Love, Huber, and Anders 2014). We grouped all 12 replicates corresponding to young flies and all 12 replicates corresponding to old flies to identify microRNAs differentially expressed independent of time of day. MicroRNAs with a Benjamini-Hochberg adjusted p-value ≤ 0.05 were considered significantly differentially expressed.\n\nWe used the command-line tool TargetScan (TargetScanFly version 7.2) (Agarwal et al. 2018) to predict mRNA targets of all microRNAs. We filtered the predicted target transcripts by expression, including only those passing a threshold of 1 FPKM (Fragments per Kilobase per Million) in our around-the-clock dataset (Kuintzle et al. 2017). For the novel small RNA analysis, small RNAs with identical seed sequences were grouped into families, and TargetScan was used to predict target transcripts as described above.\n\nWe filtered DE microRNAs to include only those with a log2 fold change ≥ 0.58 (corresponding to a simple fold change of 1.5) and expression levels ≥ 10 aRPMMM. We filtered predicted target transcripts of DE microRNAs to include only those with expression levels ≥ 10 FPKM and a probability of conserved targeting score (PCT) ≥ 0.80. PCT is calculated by TargetScanFly to provide a measure of the conservation of a microRNA site in a target transcript sequence (Friedman et al. 2009). We performed pathway analysis using the DAVID functional clustering and annotation webtool (DAVID 2021 update) with default parameters (Huang et al. 2007; Huang da, Sherman, and Lempicki 2009).\n\nTo determine if predicted microRNA target transcripts show evidence of post-transcriptional regulation, we performed exon intron split analysis (EISA) (Gaidatzis et al. 2015), examining the ratio of changes in exonic reads to changes in intronic reads between young and old flies for each transcript in our diurnal transcriptomic dataset (Kuintzle et al. 2017). We quantified intronic reads using bowtie to align FASTQ files to the Drosophila genome in an ungapped fashion using the parameters “--best --strata -m1”, then mapped the resulting reads to a FASTA file containing intronic reads downloaded from Flybase. We quantified exonic reads using bowtie to align FASTQ files to a FASTA file containing spliced transcript sequences using the parameters “-S --best --strata --all”, then mapped the resulting reads back to the spliced transcript FASTA file (also downloaded from Flybase). For each age, we used the sum of counts across all time points and replicates for exonic and intronic reads in this analysis.\n\nWe calculated an EISA score for each transcript in our dataset:\n\nWe identified novel microRNAs using the random-forest-based novel microRNA identification program miRWoods (Bell et al. 2019). For each age, we removed novel microRNA primary transcripts with only one predicted product (keeping only primary transcripts with both 5' and 3' products), and retained only the mature products (5' and 3' arms) with median expression ≥ 1 aRPMMM. The GFF files from miRWoods output were used for quantification of novel microRNAs and are included in the Underlying data, Supplementary Files 11 and 12.\n\nRNA extracted from young and old flies collected at different time points was used for reverse transcription of each sample. cDNA synthesis was achieved with the TaqMan MicroRNA Reverse Transcription Kit (Thermo Fisher, Catalog number: 4366596), used along with TaqMan Fast Advanced Master Mix (Thermo Fisher, Catalog number: 4444556) and the TaqMan™ microRNA assay (Thermo Fisher, Catalog number: 4440886) specific to miR-193-5p (Thermo Fisher assay ID 463359) and 2S rRNA (Thermo Fisher assay ID 001766), used as housekeeping reference gene.\n\nFor each biological replicate, equal amounts of cDNA from each time point were mixed to produce samples that were averaged across time points.\n\nReal-time PCR was performed with Power SYBR Green PCR Master Mix (Thermo Fisher, Catalog number: 4367659) on a QuantStudio 3 Real-Time qPCR System (Applied Biosystems) according to the manufacter’s instructions for the TaqMan small RNA assays. Relative expression was calculated with the 2-ΔΔCT method, using 2S rRNA as the reference small RNA. Expression of 2S rRNA was compared across time points and ages in both the sequencing and qPCR analyses and determined to be the best candidate for a reference small RNA, due to its constant expression across both time of day and age.\n\nMicroRNA expression plots were produced using a custom Python script (Fey 2022). Expression plots may be generated and downloaded using this webtool.\n\n\nResults\n\nFlies were collected at 5 days (young) or 55 days (old) of age, around-the-clock every 4 hours beginning at lights-on at Zeitgeber time 0 (ZT0). For each sample, 400 heads were pooled and extracted RNA was submitted for sequencing. Two biological replicates were used for each time point in both young and old ages.\n\nWe aligned processed reads to the Drosophila genome and quantified microRNA expression by counting reads overlapping mature product annotations from the miRBase version 22 GFF file (Kozomara, Birgaoanu, and Griffiths-Jones 2019). Reads were normalized relative to each million reads mapping to microRNAs, and expression values are reported in adjusted Reads per Million Mapped MicroRNAs (aRPMMM). We quantified piRNA expression at the cluster level by mapping reads to the cluster reference from the piRNA database piRNAdb version 1.7.6 and normalized relative to each million reads mapping to the genome (adjusted Reads per Million, aRPM). For novel small RNAs, reads were quantified by referencing the genomic coordinates in the GFF files produced by miRWoods (Bell et al. 2019) and normalized relative to each million reads mapping to the genome (adjusted Reads per Million, aRPM). MicroRNA expression values are available in the Underlying data, Supplementary File 1 (Hendrix and Fey 2022).\n\nGene expression profiles exhibiting diurnal rhythmicity are commonly used to identify genes regulated by the circadian clock, or directly or indirectly activated by light. We detected rhythmic microRNAs in this dataset using the RP24 method, a Fourier-based approach previously developed by our lab that measures the relative power of the 24-hour period compared to all other periods (Sebastian et al. 2022). A more positive score indicates a higher level of 24-hour rhythmicity. Briefly, we calculated the RP24 score for each microRNA passing an expression threshold of 1 aRPMMM, and considered microRNAs with a q-value ≤ 0.05 to be significantly rhythmic.\n\nOur previous work found a statistically significant net increase in transcript rhythmicity with age by comparing transcript RP24 distributions between young and old flies (Sebastian et al. 2022). To examine net changes in microRNA rhythmicity with age, we compared microRNA RP24 value distributions in young and old flies. In contrast to our previous findings for transcript rhythmicity, there was no statistically significant difference in microRNA RP24 distributions between young and old flies, suggesting that net microRNA rhythmicity is unchanged during aging (Underlying data, Supplementary Figure 1). In addition, we directly compared transcript and microRNA RP24 distributions for young and old flies and found that transcripts are significantly more rhythmic than microRNAs in both ages (Figure 2A).\n\nWe next investigated individual microRNAs with significant diurnal rhythmicity. To characterize age-related changes in rhythmicity, we grouped rhythmic microRNAs into three categories: 1) microRNAs rhythmic in young flies and arrhythmic in old flies, 2) microRNAs arrhythmic in young flies and rhythmic in old flies, and 3) microRNAs rhythmic in both ages. We identified only 19 rhythmic microRNAs: five that lost rhythmicity with age, and 11 that gained rhythmicity with age, and three that maintained rhythmicity throughout the lifespan (Figure 2B).\n\nAmong the five microRNAs that are rhythmic in young flies and arrhythmic in old flies were miR-277-5p, which is involved in branched chain amino acid catabolism and lifespan determination (Esslinger et al. 2013), and miR-92a-3p, which has been shown to cycle in PDF neurons (Chen and Rosbash 2017). Expression profiles for these examples are shown in the Underlying data, Supplementary Figure 2.\n\nThe three microRNAs rhythmic in both young and old flies were miR-92b-3p, miR-263a-3p, and miR-275-5p. Of these, miR-263a-3p, also known as bereft (bft), has previously been shown to be rhythmic and under the control of the circadian clock in young flies (Yang et al. 2008). Expression of these three examples are shown in the Underlying data, Supplementary Figure 3.\n\nMicroRNAs that gain rhythmicity in old flies included miR-33-3p and miR-34-5p, which has previously been shown to be strongly upregulated with age in Drosophila brains (Liu et al. 2012). Expression profiles of these examples are shown in the Underlying data, Supplementary Figure 4.\n\nSurprisingly, we found no microRNAs with statistically significant rhythmicity for both 5' and 3' products. Based on the microRNA transcription process (Figure 1A), rhythmic transcription of the microRNA primary transcript should result in rhythmic expression for both mature products; however, it has been shown that while the non-functional miR* is degraded in the cell, the functional miR is stabilized by association with Ago1 (Van den Brande et al. 2018). We hypothesized that incorporation into the RISC and subsequent stabilization may mask temporal expression patterns for functional miRs, which remain visible for the non-stabilized miR*. To test this hypothesis, we used relative expression levels to categorize rhythmic microRNAs as highly-expressed functional miRs or lowly-expressed miR* products. We found that eight out of the 19 rhythmic microRNAs are miR* products: miR-277-5p, miR-275-5p, miR-1003-5p, miR-12-3p, miR-1007-5p, miR-33-3p, miR-87-5p and miR-263a-3p. We visualized the expression of the rhythmic miR* miR-263-3p and the corresponding non-rhythmic miR-263a-5p in both young (Figure 2C) and old (Figure 2D) flies. We suggest examination of such miR* expression profiles may offer insights into circadian or light-activated microRNA regulation which is invisible in functional miR expression profiles.\n\nWe also identified diurnally-expressed piRNAs using the RP24 method and found only one significantly rhythmic cluster, which was arrhythmic in young flies and rhythmic in old flies (Underlying data, Supplementary Figure 5). We visualized this piRNA cluster on the Broad Institute’s Integrative Genomic Viewer (IGV) tool (Figure 2E) (Thorvaldsdottir, Robinson, and Mesirov 2013), and found that this cluster overlaps RpL3, encoding a ribosomal protein, as well as three noncoding RNAs: two small nucleolar RNAs hosted in RpL3, and the antisense RNA CR31144 located on the opposite strand. The full lists of rhythmic microRNAs are available in the Underlying data, Supplementary File 2.\n\nBecause we detected relatively few rhythmic microRNAs and piRNA clusters, we deemed it important to conduct age-dependent but time-of-day-independent analyses. To investigate global changes in small RNA expression with age, we examined the number of reads mapped to annotated microRNAs at all time points, separately for young and old flies, normalized relative to the total number of reads mapped to the genome (Figure 3A). We find that the number of microRNA reads is significantly lower in old flies (p-value < 0.001, t-test), indicating that global microRNA expression decreases with age.\n\nGlobal total RNA expression decreases during aging in Drosophila (Davie et al. 2018); therefore, we tested whether the observed global decrease in microRNA expression was due simply to a lower number of genome-mapped reads in old flies. Surprisingly, examination of the total number of reads mapped to the genome in both ages revealed a significantly higher number in old flies (p-value < 0.001, t-test, Figure 3B). This suggests that there is an upregulation in old flies of an RNA product with a similar size range as microRNAs. The read length distribution for all samples in young flies shows a strong, sharp peak at 22 nucleotides (nt) (Figure 3C), corresponding to the size of Drosophila microRNAs (Xue and Zhang 2018). In old flies, this 22-nt peak is severely dampened; however, there is an increase in genome-mapped reads from 24 to 29 nt, corresponding to the size range of piRNAs (Brennecke et al. 2007).\n\nWe hypothesized that the increase in genome-mapped reads in old flies is due to an age-associated upregulation in the expression of piRNAs, small RNAs that target transposons. Transposon expression and activity is known to increase during aging in fly heads (Li et al. 2013); therefore, piRNA expression may also increase as a protective measure. To test this hypothesis, we quantified piRNA reads and examined the number of reads mapped to piRNA clusters for young and old flies, normalized relative to the total number of genome-mapped reads (Underlying data, Supplementary Figure 6). We find that more reads map sense to piRNA clusters in old flies compared to young flies, suggesting an increase in global piRNA expression during aging. Interestingly, we also find an increase in reads mapping antisense to piRNAs. These reads may be derived from transposons, which are used as templates to produce piRNAs (Czech and Hannon 2016), suggesting that transposon expression may be increased in old flies in this dataset.\n\nOur previous work identified five putative piRNA primary transcripts with age-induced rhythmicity (Kuintzle et al. 2017). We confirmed expression of small RNA reads deriving from these five primary transcripts in this dataset, with two expressed at levels ≥ 1 aRPM in at least one age (Underlying data, Supplementary Figure 7). The piRNA expression data and primary transcript genomic coordinates are available in the Underlying data, Supplementary Files 3 and 4.\n\nTo understand how the expression of individual microRNAs is affected in the heads of aging flies, we performed differential expression analysis using two R packages, DESeq2 (Love, Huber, and Anders 2014) and edgeR (Robinson and Oshlack 2010). We defined a high-confidence set of 170 differentially expressed (DE) microRNAs which were identified as significantly DE by both programs (Underlying data, Supplementary Figure 8). We found that 68 microRNAs were significantly upregulated and 102 were significantly downregulated in old flies compared to young.\n\nWe examined DE microRNAs which passed a median expression threshold of 1 aRPMMM in either age in Figure 4A, where labeled microRNAs have expression ≥ 5 aRPMMM. The most highly upregulated microRNA was the miR-2 family member miR-13b-2-5p, with the miR-2 family member miR-13a-5p also significantly upregulated with age. The microRNA miR-308-3p, which targets Myc, an ortholog to a human oncogene controlling cell growth and proliferation (Daneshvar et al. 2013), was also significantly higher in old flies compared to young.\n\nThe most strongly downregulated microRNA in old flies compared to young was miR-193-5p, which has been implicated in the immune response in young flies (Atilano et al. 2017). We confirmed dramatic decrease in expression levels for miR-193-5p using RT-qPCR (Figure 4B). Also downregulated with age were miR-993-3p and miR-990-5p, the latter of which acts in glial cells to regulate circadian locomotor activity (You et al. 2018). Another member of the large multi-cluster miR-2 family, miR-2c-5p, was also downregulated in old flies.\n\nThe differential expression of various miR-2 microRNAs prompted us to investigate this family further. The miR-2 family of microRNAs is well-conserved in invertebrates: the 3' arm of microRNAs belonging to this family is very highly conserved, and is generally acknowledged as the functional miR, while the 5' miR* arm is more variable in sequence (Marco, Hooks, and Griffiths-Jones 2012). We observed no upregulation of 3' products; however, five 5' products were upregulated in old flies (Figure 4C). We also note a surprising inconsistency in expression changes with age for three clustered miR-2 family members (miR-2c, miR-13a and miR-13b-1), which are hosted in the long non-coding RNA (lncRNA) CR45911 and are thought to be under common transcriptional control. Both miR-2c-3p and miR-2c-5p are downregulated, while miR-13a-5p is upregulated in old flies. The remaining members of this cluster, miR-13a-3p, miR-13b-3p and miR-13b-1-5p, were not significantly differentially expressed in our data (Figure 4C). This difference in age-related expression changes among clustered miR-2 family members hints at an age-onset change in stability or processing for individual microRNAs.\n\nWe also performed differential expression analysis to identify age-dependent changes in piRNA cluster expression. We defined a high-confidence set of DE piRNAs which were detected with both edgeR (Robinson and Oshlack 2010) and DESeq2 (Love, Huber, and Anders 2014). We found two piRNA clusters significantly upregulated and two significantly downregulated with age (Underlying data, Supplementary Figure 9). Notably, the downregulated piRNAdb Cluster 44 (Figure 4D), which contains 16 individual piRNAs, overlaps the well-studied flamenco locus. The flamenco lncRNA hosts the largest piRNA cluster in Drosophila somatic cells, and acts to repress gypsy transposon activity (Ozata et al. 2019). This age-dependent downregulation of a proven piRNA-producing locus may partially explain observed increases in gypsy transposon expression and activity with age (Li et al. 2013). Full lists of DE small RNAs identified by each DE analysis program are available in the Underlying data, Supplementary Files 5 through 8.\n\nTo explore the potential biological impacts of the most highly DE microRNAs in our dataset, we first integrated a transcriptomics dataset previously generated by our lab (Kuintzle et al. 2017), then performed pathway analysis on the predicted targets of DE microRNAs. We filtered DE microRNAs to include those with absolute log2 fold change values ≥ 0.58 (corresponding to a simple fold change of 1.5) and expression values ≥ 10 aRPMMM, resulting in 21 downregulated and 13 upregulated microRNAs. We used the target prediction tool TargetScanFly version 7.2 (Agarwal et al. 2018) to predict target transcripts of each DE microRNA, then filtered the transcripts by expression and probability of conserved targeting score (see Methods). With these filters, downregulated microRNAs had 76 unique predicted target transcripts, and upregulated microRNAs had 174 unique predicted target transcripts. We used the DAVID functional annotation webtool (Huang et al. 2007; Huang da, Sherman, and Lempicki 2009) to identify functional pathways that may undergo changes in post-transcriptional regulation with age due to the differential expression of microRNAs.\n\nWe visualized the log2 fold change values for transcripts in each significant cluster resulting from analysis of predicted targets of upregulated (Figure 5A) and downregulated (Figure 5B) microRNAs. We noted that the transcripts belonging to these enriched pathways have low to moderate log2 fold change values. In some cases, transcript expression is altered in the same direction as the expression of the DE microRNAs predicted to target them. While this may seem counterintuitive at first glance, post-transcriptional regulation functions in concert with other determinants of expression level, such as basal transcription rate; therefore, change in expression level alone cannot be the sole evidence for microRNA targeting. In addition, it has been found that most microRNAs cause only modest changes in target expression, and in a system-wide context microRNAs are thought to act as buffers that protect against large changes in transcript expression for important pathways (Bartel 2018; Ebert and Sharp 2012). Therefore, targets of DE microRNAs may be post-transcriptionally regulated even though a large change in transcript expression with age is not observed.\n\nWe investigated pathways associated with targets of upregulated microRNAs (Figure 5A) and found that the largest and most highly enriched pathway contained transcripts encoding membrane proteins, including Rh7-RA, a rhodopsin photoreceptor playing a role in circadian entrainment to light, and Nlg3-RC, a neuroligin involved in synaptic transmission. Predicted target transcripts also encoded transcription factors, including several related to stress and aging. Among these were Mnn1-RC, which regulates multiple stress response pathways, including hypoxia and oxidative stress, and REPTOR-RB (Repressed by TOR), which is involved in regulating stress and aging with TOR (target of rapamycin). Other predicted targets of upregulated microRNAs were involved in synaptic growth at the neuromuscular junction, and encoded proteins containing immunoglobulin, zinc finger, and leucine-rich repeat domains.\n\nWe also explored pathways associated with predicted targets of downregulated microRNAs (Figure 5B). We found that the most highly enriched group of transcripts encoded mRNA binding proteins, including elav-RD, which is neuronally expressed, and msi-RF, which encodes a protein that represses the hypoxia inducible factor (HIF) pathway by targeting the 3' UTRs of mRNAs. One of the genes targeted by Msi is the transcription factor ttk (tramtrack), which has been proposed as a putative regulator of pdf, the main circadian neuropeptide in Drosophila (Mezan et al. 2016). Also included in this group was mbf1-RB, which encodes a transcriptional co-activator that induces transcription of stress response genes, and has one of the highest log2 fold changes of transcripts in this cluster. Other transcripts predicted to be targets of significantly downregulated microRNAs were involved in calcium-ion binding and intracellular signal transduction, and encoded membrane and developmental proteins. Full pathway analysis results are available in the Underlying data, Supplementary File 9.\n\nNext, we refined our exploration of the potential biological effects of age-related microRNA expression changes on predicted target transcripts. We used a previously published method, exon intron split analysis (EISA), to determine the extent to which a transcript is likely undergoing changes in post-transcriptional regulation with age (Gaidatzis et al. 2015). For every transcript we calculated an EISA score:\n\nWe found that 98 transcripts had a significant EISA score, indicating that they are likely experiencing age-related changes in post-transcriptional regulation (Figure 6A). We found 38 transcripts with a positive score, and 60 with a negative score, suggesting that over one-and-a-half times as many transcripts experience increased rather than decreased post-transcriptional regulation in old flies according to this score. We noted that many of these transcripts do not show strong expression differences between young and old flies, highlighting the usefulness of this score for uncovering evidence of post-transcriptional regulatory changes with age.\n\nWe next investigated which microRNAs might be responsible for mediating these changes in post-transcriptional regulation with age. We focused our analysis on significant transcripts that have predicted binding sites for DE microRNAs that pass a median expression threshold of 1 aRPMMM in either age. Next, we ensured that the calculated transcript EISA score was anti-correlated with the log2 fold change of the DE microRNA; that is, we kept transcripts with negative EISA scores predicted to be targeted by upregulated microRNAs, and transcripts with positive EISA score predicted to be targeted by downregulated microRNAs (pairs available in the Underlying data, Supplementary File 10). We found that 79 significant transcripts had predicted binding sites for DE microRNAs with anti-correlated log2 fold change values (Underlying data, Supplementary Figure 10).\n\nWe examined the strongest microRNA-transcript pairs: microRNAs with absolute log2 fold change values ≥ 0.58 (corresponding to a simple fold change of 1.5) and predicted targets with EISA scores ≥ 1.5 or ≤ -1.5. We found 19 transcripts corresponding to 15 genes passing these thresholds. Interestingly, 10 of these transcripts have demonstrated roles in the maintenance or regulation of muscle.\n\nWe first examined transcripts showing evidence of increased post-transcriptional regulation with age (Figure 6B). The transcript in this group with the strongest EISA score was Boot-RA, which is involved in the nuclear export of piRNAs (ElMaghraby et al. 2019). We also found eight transcripts known to be involved in muscle function: SERCA-RA, Mlp60A-RE, Mlp84B-RA, Mlp84B-RB, Mlp84B-RC, Prm-RE, Act-87E-RA, and Nlg1-RD. SERCA-RA encodes an endoplasmic reticulum calcium pump, and Mlp60A-RE encodes a muscle LIM-domain protein which maintains flight muscles; both have been implicated in the molecular mechanism underlying aging muscle (Bordet et al. 2021; Delrio-Lorenzo et al. 2020). Mlp84B is another muscle LIM protein with roles in muscle maintenance (Clark, Bland, and Beckerle 2007). An additional transcript in this group was Nlg1-RD (Neuroligin 1), encoding an adhesion protein that plays a role in the function of postsynaptic neuromuscular junctions (Gaudet et al. 2011; Banerjee, Venkatesan, and Bhat 2017; Owald et al. 2012). Lastly, we found Prm-RE, encoding an invertebrate-specific muscle protein (Becker et al. 1992), and Act87E-RA, an actin with diverse roles, including in muscle contraction (Roper, Mao, and Brown 2005).\n\nWe found that many of these muscle-related transcripts are predicted targets of a small subset of microRNAs, including miR-9a-5p, which plays diverse roles in development, including regulating the formation of the junction between muscle and tendons (Yatsenko and Shcherbata 2014). It is predicted to target five of these eight transcripts. Several of these microRNAs have been shown to be expressed in Drosophila thoracic muscle (Fulga et al. 2015), including the circadian-associated let-7-5p, which is predicted to target five of these transcripts, and the circadian-regulated miR-263a-5p, which is predicted to target Act87E-RA and Nlg-RD. Mlp84B-RC and Act87E-RA are predicted targets of miR-13a-5p, which is also expressed in thoracic muscle. Expression of a miR-13a sponge in thoracic muscle results in flies with impaired flight phenotypes (Fulga et al. 2015).\n\nWe also examined transcripts showing evidence of decreased post-transcriptional regulation with age (Figure 6C). These included Tm2-RB and Tm2-RG, encoding tropomyosin 2, which is critical for regulating calcium-dependent muscle contraction. Both isoforms have predicted binding sites for downregulated miR-133-5p, a conserved microRNA that regulates muscle development in mice and Xenopus (Boutz et al. 2007; Chen et al. 2006). This group also included Rnmt-RA, which encodes a methyltransferase that caps mRNAs. Taken together, these results suggest the altered expression of microRNAs potentially involved in muscle-regulatory networks during aging.\n\nWe used the random-forest-based microRNA detection program miRWoods (Bell et al. 2019) with the goal of identifying novel microRNAs in our dataset. We ran the program separately on data from young and old flies, and identified 121 novel small RNA primary transcripts between the two ages. We retained primary transcripts which had two predicted mature products (5' and 3' arms), resulting in a high-confidence set of 58 novel small RNA primary transcripts. We filtered the corresponding mature products by expression level and retained 24 novel mature small RNA products: four were detected only in young flies, eight were detected in both ages, and 12 were detected only in old flies (Figure 7A).\n\nWe used TargetScanFly (Agarwal et al. 2018) to predict target transcripts for each novel small RNA family (see Methods), then filtered the results to include only transcripts expressed at levels ≥ 1 FPKM in our dataset. We found that most of the newly identified small RNAs are predicted to target between 400 and 2000 transcripts, while many previously characterized microRNAs are predicted to target appreciably more transcripts (Figure 7B).\n\nWe performed differential expression analysis (as described above) to characterize age-associated expression changes for each novel discovery (Figure 7C). We found that 19 out of the 24 novel small RNAs were differentially expressed with age: 16 were upregulated in old flies, and three were downregulated in old flies.\n\nWe also performed rhythmicity detection using the RP24 score to identify small RNAs with 24-hour periodicity that may be regulated by light or by the circadian clock. We found six rhythmic small RNAs, all of which were arrhythmic in young flies, but gained rhythmicity in old flies, with peak expression at ZT16 (Figure 8A). Notably, these six small RNAs identified as rhythmic in old flies were all identified as significantly upregulated with age.\n\nSurprisingly, we found that these six rhythmic predicted microRNAs were novel transfer RNA derived fragments (tRFs). These recently discovered small molecules are hosted in mature or precursor tRNA sequences, and are classed according to the region of the tRNA from which they derive (Kumar et al. 2015; Krishna et al. 2021). Two classes map to mature tRNAs: tRF-5s map to the 5' end, and tRF-3s map to the 3' end of mature tRNAs. Those deriving from tRNA precursor sequences are named tRF-1s. Previous studies have observed tRFs in bacteria, plants, and animals, including Drosophila, mouse, and humans (Krishna et al. 2021).\n\nWe observed that the expression profiles for the six rhythmic tRFs overlap, and that they are all 5' products (Figure 8A). We examined expression profiles of their corresponding 3' products and found that these also overlap (Figure 8B), suggesting a high sequence similarity among 5' and among 3' products. Indeed, we found that all six of the 5' products share an identical 26-nt sequence, while the corresponding six 3' products share an identical 24-nt sequence. Further investigation revealed that the primary transcript sequences for five of these products are also identical, resulting in identical predicted dot-bracket structures, which we visualized using the RNA structure visualization tool VARNA (Figure 8C). The sequence of the sixth tRF primary transcript differs slightly, producing a different predicted structure (Underlying data, Supplementary Figure 11A). We conclude that these tRF loci exhibit microRNA-like characteristics, because miRWoods uses a random forest trained on known microRNA features, and we hypothesize that the predicted hairpin structure of the tRF primary transcripts contributed to the identification of these products with miRWoods (Bell et al. 2019).\n\nWe visualized the log-transformed reads for these tRFs using IGV (Figure 8D) and found that five of the six primary transcripts are clustered on either side of the uncharacterized gene CG8490. The sixth primary transcript is located on the same chromosome (2R), approximately 1000 base pairs downstream of the long noncoding RNA (lncRNA) CR44472. Visualization on the IGV tool confirmed that five of the primary transcripts map to loci encoding GTG Histidine transfer RNAs (tRNAs), and the sixth maps to tRNA:His-GTG-2-1Ψ-RA, a pseudo-tRNA (Figure 8D and Underling data, Supplementary Figure 11B).\n\nThe alignment patterns of these pairs of 5' and 3' products to tRNAs suggests that they are pairs of tRF-5 and tRF-3 molecules. We searched the tRF database tRFdb (Kumar et al. 2015) for these tRFs using genomic coordinates and sequences, and concluded that these are not currently annotated as tRFs. Thus, we report the discovery of novel tRFs which show age-onset expression and rhythmicity.\n\n\nDiscussion\n\nHere we present a systems-level exploration of diurnal changes in small RNA expression with age, including microRNAs, piRNAs, and the newly-discovered class of small RNAs, transfer RNA derived fragments (tRFs). We report an age-related increase in microRNA rhythmicity, but a global decrease in microRNA expression with age, corroborating results from studies in C. elegans, mice and humans (Ibanez-Ventoso et al. 2006; Inukai et al. 2012; Noren Hooten et al. 2010), and show a corresponding increase in global piRNA expression levels with age, suggesting a shift in the regulatory small RNA profile of Drosophila during aging.\n\nAn important contribution from our study is the identification of novel tRFs using the random-forest-based microRNA detection program miRWoods (Bell et al. 2019). Similar to microRNAs, tRFs have seed sequences which match transcript 3' UTR regions conserved in Drosophilids (Lee et al. 2009; Karaiskos et al. 2015). Additionally, they are loaded into Ago1 and Ago2 in Drosophila, and in some cases, they have been shown to silence target transcripts (Krishna et al. 2021; Karaiskos et al. 2015).\n\nWe found 12 novel tRFs (six tRF-5s and six tRF-3s) hosted in five tRNAs and one pseudo-tRNA. All 12 are upregulated in old flies, and the six tRF-5s are rhythmic in old flies but not in young, hinting at an age-associated regulatory role for these newly-discovered tRFs. While tRFs have been shown to be age-induced in Drosophila (Karaiskos et al. 2015), the characterization of 24-hour rhythmic expression patterns is a novel finding. Notably, we do not observe the canonical ‘CCA’ trinucleotide for the novel tRFs mapping to the 3' end of the mature tRNAs, suggesting that these tRF-3s derive from tRNAs which have not been fully processed into mature products. There is evidence for age-dependent changes in expression and activity of some tRNA modifying enzymes (Zhou et al. 2021); it is possible that changes in these modifications may alter the way in which tRNAs fold into their classic functional tertiary structure. Further, the program miRWoods identifies novel microRNAs based on the similarity of primary transcript sequences to a hairpin structure. This suggests that in the absence of chemical modifications required for proper folding, these tRNA molecules resemble a primary microRNA transcript. It is possible that age-related changes in tRNA chemical modifications affect their processing, allowing for the biogenesis and expression of tRFs in old flies.\n\nWe identified several differentially expressed microRNAs which have previously been shown to have neuroprotective roles in aging Drosophila. These include the downregulated miR-1000-5p, which plays a neuroprotective role in light-dependent regulation of presynaptic glutamate release (Verma et al. 2015), and the upregulated miR-263a-3p, which similarly regulates glutamate excitotoxicity, specifically in glia (Aw et al. 2017).\n\nSeveral microRNAs with predicted roles in immunity were downregulated with age in our data, including miR-1004-3p, which is predicted to target puc (puckered), a serine/threonine phosphatase involved in the Jun-N-terminal kinase (JNK) pathway (Fullaondo and Lee 2012), and miR-193-5p. The conserved miR-193-5p is involved in immune and stress response pathways in young flies and may play a role in buffering the response to infection (Atilano et al. 2017).\n\nWe found relatively few microRNAs with significant 24-hour periodicity, similar to previous microarray studies of diurnal microRNA expression in young flies (Yang et al. 2008; Vodala et al. 2012). Yang et al. showed miR-263a to be rhythmically expressed under the control of the circadian clock in young flies (Yang et al. 2008). This microRNA is part of the miR-263 family conserved in mammals, and has been shown to be expressed in Drosophila brains (Liu et al. 2012). We found that miR-263a-3p is upregulated with age, and we corroborate the study by Yang et al. showing that it is rhythmic in young flies. Further, we show that it maintains rhythmicity in old age.\n\nWe note that several miR* products are rhythmic in our dataset, while the corresponding functional miR is arrhythmic. Importantly, it has been shown that the functional miR is protected from degradation by binding to the stable Ago1 in Drosophila (Van den Brande et al. 2018). We propose a scenario in which the association of the functional miR with Ago1 may mask temporal expression patterns that are observable in the miR*. It has previously been shown that most circadian-regulated microRNAs in mouse liver are arrhythmic at the level of the mature microRNA product, but may be identified by the oscillations of the primary transcript (Wang et al. 2016). In a similar vein, we suggest that identification of oscillating miR* products may shed light on underlying regulation of microRNAs by light or by the circadian clock. We showed one example of a microRNAs with an arrhythmic functional miR and a rhythmic miR* products: the circadian-regulated miR-263a. Another example is miR-33-3p, which gains rhythmicity with age. When depleted in Drosophila glia, miR-33 affects rhythmic behavior, suggesting that it is involved in circadian regulation (You et al. 2018). Thus, we further suggest that age-related changes in circadian or light-activated regulation may be deduced by examining changes in miR* rhythmicity between young and old flies.\n\nAnother key finding of this study is the evidence for the age-induced increase in post-transcriptional regulation of transcripts involved in muscle maintenance and development. Our transcriptomic data are derived from whole heads, which contain several muscle groups operating the proboscis during feeding (McKellar et al. 2020). Several differentially expressed microRNAs are predicted to target these muscle-related transcripts. Transcripts showing evidence of decreased post-transcriptional regulation with age are predicted to be targets of the downregulated conserved miR-133-5p, which is muscle-associated in Xenopus and in mouse myoblasts (Chen et al. 2006; Boutz et al. 2007). The upregulated circadian-associated microRNAs let-7-5p and miR-263a-5p, both of which have been shown to be expressed in muscle tissue in Drosophila (Fulga et al. 2015), are predicted to target several transcripts that show evidence of increased post-transcriptional regulation with age. In addition, these transcripts are predicted targets of the upregulated miR-9a-5p, a regulator of muscle-tendon junction development (Yatsenko and Shcherbata 2014). Among these are all three isoforms of the gene Mlp84B, which has been shown to be downregulated with age in whole Drosophila (Bordet et al. 2021). Interestingly, we find that the most highly expressed isoform of Mlp84B, Mlp84B-RA, is upregulated in old flies in our transcriptomics dataset. This difference may stem from tissue-specific expression patterns for this transcript. We note that the use of whole fly heads prevents us from drawing definite conclusions about the potential for microRNA targeting of specific transcripts because we do not know that both the microRNA and its target are expressed in the same cell. However, these results hint at an age-onset reprogramming of muscle regulatory networks and suggests that a limited set of microRNAs may play a large role in regulating muscle during the aging process. We propose these microRNA-target pairs for future experimental analyses to confirm expression in the same cell and tissue types, prior to validation of targeting and biological impact.\n\nThe insights gained from this characterization of diurnal small RNAs in both young and old Drosophila advance our understanding of age-related changes in small regulatory molecule expression, and set the stage for continued exploration of the role of these players in aging circadian systems.",
"appendix": "Data availability\n\nOpen Science Framework: small RNA diurnal expression Drosophila.\n\nhttps://doi.org/10.17605/OSF.IO/KVP2Q (Hendrix and Fey 2022).\n\nThis project contains the following underlying data:\n\n• README.docx (Supplementary Figure captions and Supplementary File descriptions)\n\n• SupplementaryFigure10_EISA.pdf (Scatter plots of anti-correlated pairs of DE microRNAs and predicted transcripts.)\n\n• SupplementaryFigure11_rr817650-3a.pdf (Unclustered novel tRF.)\n\n• SupplementaryFigure1_histRP24s_youngVsOld_mirs.pdf (Histogram of RP24 value distributions of microRNAs.)\n\n• SupplementaryFigure2_R2A.pdf (Expression profiles for two microRNAs rhythmic in young flies and arrhythmic in old flies.)\n\n• SupplementaryFigure3_R2R.pdf (Expression profiles for three microRNAs rhythmic in both young and old flies.)\n\n• SupplementaryFigure4_A2R.pdf (Expression profiles for two microRNAs arrhythmic in young flies and rhythmic in old flies.)\n\n• SupplementaryFigure5_Cluster21.pdf (Expression profiles for piRNA cluster 21.)\n\n• SupplementaryFigure6_piRNAs.pdf (Global piRNA expression.)\n\n• SupplementaryFigure7_putative_piRNAs.pdf (Putative piRNA expression profiles.)\n\n• SupplementaryFigure8_DEmirs_noNovel.pdf (Differentially expressed microRNAs.)\n\n• SupplementaryFigure9_barplot_DE_piRNAs_DESeq_edited.pdf (Differentially expressed piRNA clusters.)\n\n• SupplementaryFile10_anticorrelatedEISA.xlsx (Pairs of differentially expressed microRNAs and their predicted target transcripts with significant EISA scores.)\n\n• SupplementaryFile11_miRWoodsPredictions_youngNovel.gff (GFF file with genomic information for novel microRNAs run on data from young flies.)\n\n• SupplementaryFile12_miRWoodsPredictions_oldNovel.gff (GFF file with genomic information for novel microRNAs run on data from old flies.)\n\n• SupplementaryFile1_expression_mirs_known.xlsx (Normalized microRNA expression levels.)\n\n• SupplementaryFile2_rhythmicMirs.xlsx (MicroRNAs with statistically significant rhythmicity.)\n\n• SupplementaryFile3_putative_piRNA_expression.xlsx (Normalized putative piRNA expression levels.)\n\n• SupplementaryFile4_putative_piRNA_transcript_coordinates.gtf (GTF file with putative piRNA primary transcript coordinates.)\n\n• SupplementaryFile5_DESeq2_results_mirs_known.xlsx (Differentially expressed microRNAs according to DESeq2.)\n\n• SupplementaryFile6_edgeR_results_mirs_known.xlsx (Differentially expressed microRNAs according to edgeR.)\n\n• SupplementaryFile7_DESeq2_results_piRNAs.xlsx (Differentially expressed piRNAs according to DESeq2.)\n\n• SupplementaryFile8_edgeR_results_piRNAs.xlsx (Differentially expressed piRNAs according to edgeR.)\n\n• SupplementaryFile9_DAVIDresults.xlsx (DAVID pathway analysis results.)\n\nData are available under the terms of the Creative Commons Zero \"No rights reserved\" data waiver (CC0 1.0 Public domain dedication).\n\nAccession number\n\nNCBI GEO: Diurnal small RNA expression and post-transcriptional regulation in young and old Drosophila melanogaster heads. Accession number: GSE210559; https://identifiers.org/geo:GSE210559\n\nRaw and processed data has been deposited to NCBI Gene Expression Omnibus (GEO) under accession number GSE210559.\n\nAnalysis code available from: https://github.com/rfey/small-RNA-aging-diurnal-transcriptome\n\nArchived analysis code at time of publication: https://doi.org/10.5281/zenodo.7083424 (Fey 2022)\n\nLicense: GNU General Public License v3.0\n\n\nAcknowledgements\n\nWe are grateful to Mark Dasenko for peparing small RNA libraries and perfoming sequencing. We thank Jimmy Bell for running the miRWoods analysis.\n\n\nReferences\n\nAcosta-Rodriguez VA, Rijo-Ferreira F, Green CB, et al.: Importance of circadian timing for aging and longevity. Nat. Commun. 2021; 12: 2862. PubMed Abstract | Publisher Full Text\n\nAgarwal V, Subtelny AO, Thiru P, et al.: Predicting microRNA targeting efficacy in Drosophila. Genome Biol. 2018; 19: 152. 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}
|
[
{
"id": "168339",
"date": "06 Jun 2023",
"name": "Taichiro Iki",
"expertise": [
"Reviewer Expertise Drosophila melanogaster. Gene regulation. Small RNA biogenesis."
],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address 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 'Diurnal small RNA expression and post-transcriptional regulation in young and old Drosophila melanogaster heads' performed systemic profiling of miRNAs and other small non-coding RNAs expressed in the heads of Drosophila melanogaster maintained in 12h:12h light/dark cycle. The authors prepared young (5 days) and old (55 days) conditions and compared the difference in sRNA expression pattern. The authors found a group of miRNAs showing rhythmicity. sRNA data were combined with transcriptome dataset published in their previous study and sRNA putative functions were discussed.\nFig 2CD. GSE210559 contain biological triplicates data for a certain ZT (0, 4, 8, 12, 16, 20). I couldn’t get how the authors can generate the panel like Fig. 2CD showing 48h (not 72h) scale in X-axis. Values in one time point in Fig. 2C correspond to single replicate data?\n\nFig 2E. The track view does not have any values for Y-axis. Mean RPM can be shown in bedgraph.\n\nFig 2E. The authors indicate these are “piRNAs”. But, I am not sure if these fragments are truly piRNAs or not. Other supporting evidence is necessary (for example, 5’Uridine enrich, 24-26nt size peak, PIWI interaction).\n\nFig 3AB. A decrease of miRNA per total genome mapper can be simply because of the increase of total genome mappers. The authors cannot conclude \"global microRNA expression decreases with age.\" These data say “proportion of canonical miRNAs were reduced in the analyzed pool”. RT-qPCR can measure the absolute miRNA levels.\n\nFig 3C. Please indicate in the legend if one red/blue line corresponds to a single replicate of deep-seq. Material Methods say 18-29-nt were analyzed, but panel C contains 30 and 31 nt species. Is it OK?\n\nFig. 4B. Need statistics test. I believe the most proper RT-qPCR statistics is a way not giving value 1.0 to each replicate in the control (day 5) condition. Since 2S was used for reference, delta CT can be used for t-test. In the authors’ data, biological triplicate data always show 1 (set as 1). But, in reality, the values should be different between control conditions. Using the fluctuating values, mean value can be given, and statistics can be done.\n\nFig. 4C. Please provide error bar (can be given by 12 replicates). X-axis values are log scale or linear scale? Not indicated.\n\nBootlegger (boo-RA) cannot be found in Figure 6.\n\nMain text for Fig. 6. “circadian-associated” let-7-5p is shown in this study? At least no reference is cited. What is the difference between “circadian-associated” and “circadian-regulated”?\n\nMain text for Fig. 6C part. “This group also included Rnmt-RA, which encodes a methyltransferase that caps mRNAs” does not fit to the following conclusion “microRNAs potentially involved in muscle-regulatory networks”.\n\nFig. 7A. The 24 sRNAs remained after filter of “expression level”. The threshold is not indicated.\n\nFig. 7A. Hairpin structures of precursors for 24 novel small RNAs can be shown. (Like the one in Fig. 8C)\n\nFig. 7C. The main text says “differentially expressed”. What is the threshold? Looks different from the one (log2 0.58) used in other parts.\n\nFig. 8D. Scale bar is missing for the track view. Please give a magnified view for CG8490, excluding surrounding tRNA genes.\n\nText polishing is required. Not easy to follow the story.\n\nIntroduction (Fig 1B part). “the production of secondary piRNAs using a primary piRNA as a template” is not correct. Secondary piRNAs are produced from fragments generated by primary piRNA-directed cleavage.\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nPartly\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Partly",
"responses": []
},
{
"id": "233275",
"date": "12 Feb 2024",
"name": "Alex Flynt",
"expertise": [
"Reviewer Expertise small RNA biology",
"Drosophila genetics",
"transcriptomic analysis"
],
"suggestion": "Not Approved",
"report": "Not Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe article by Fey et al. analyzes a dataset intended to compare small RNAs between young and old flies that are subject to changes in expression over a circadian cycle. The authors examine the expression of miRNAs, piRNAs, fragments of tRNAs, and possible novel small RNAs in the work. Unfortunately, as stated on page 8, there is only evidence for a handful of miRNAs that seem to differ by age. The only significant changes appear be related to passenger/miR* read abundances. The authors also examine piRNA, likewise finding very little compelling data for rhythmic expression. After this the paper begins a comparison between young and old datasets in bulk. In this they compare the overall mapping of miRNAs vs genome and find a large change. Following those analyses the article examines miRNA targets and seeks to annotate novel small RNAs.\nGiven the rudderless nature of the work that also suffers from inappropriate analyses and lack of functional experiments I recommend the article be rejected. The datasets could yield some interesting insights, but this might not be related to circadian rhythms and rather aging. The authors have some interesting observations about shifts of small RNAs in old flies. Focusing on these observations and taking advantage of the drosophila genetics toolbox for functional tests would lead to a much more impactful study. More critical to my recommendation for rejecting the manuscript is the analyses of piRNAs and novel small RNAs are extremely problematic. Issues are described in more detail below.\nMajor criticism:\nThe articles presents piRNAs in figure 2. These are not piRNAs, but rather fragments of snoRNAs. This is clear from the annotations. Small RNA sequencing is often contaminated with fragments of abundant RNAs–snoRNAs being one. To this point it is clear from the figure that neither the pingpong cycle or the phasing type piRNAs are being produced from this locus. Along these lines, like snoRNAs, tRNAs are also frequently recovered in small RNA sequencing. I suspect these degradation products may be more abundant in the old fly libraries and be the cause of the bias towards lower miRNA levels in those samples. The authors should take full advantage of the extensive genome annotations in melanogaster to understand their observation. It is not clear how the authors are quantifying coding transcripts. There is not an accession number for mRNA seq and no mention of creating these libraries in the methods or mapping of public data. Perhaps this is an oversight? but it is worrisome that the small RNA sequencing data has been used for these quantifications, and the results shown in figure 5 and 6 are not meaningful. Another large issue is the suggestion that novel miRNAs were found. There are multiple characteristics that need to be assessed such as Dicer cleavage patterns in order to confidently find a novel miRNA. The authors do not provide this information. Again my impression of the data is that there is a greater number of degradation products in the old data and the small RNA seq has more than anything served as a measure of the degradome.\n\nMinor criticism:\nFigure 1 is unnecessary. These diagrams can be found in a variety of reviews and don’t need to be rehashed here. Further, mentioning the pingpong cycle is not helpful as the authors do not look for this signature in their data. Figure 2E and 2D, points should have error bars as each seems to be the average of values from two biological replicates. Figure 4B the graphic is low quality. Figure 5 shows to be a negative result, which does little to further the narrative. Figures 6B and 6C are hard to understand, the authors should consider alternative methods of graphing their data.\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? No\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Partly\n\nAre the conclusions drawn adequately supported by the results? No",
"responses": []
}
] | 1
|
https://f1000research.com/articles/11-1543
|
https://f1000research.com/articles/11-1541/v1
|
21 Dec 22
|
{
"type": "Genome Note",
"title": "An updated version of the Madagascar periwinkle genome",
"authors": [
"Clément Cuello",
"Emily Amor Stander",
"Hans J. Jansen",
"Thomas Dugé De Bernonville",
"Audrey Oudin",
"Caroline Birer Williams",
"Arnaud Lanoue",
"Nathalie Giglioli Guivarc'h",
"Nicolas Papon",
"Ron P. Dirks",
"Michael Krogh Jensen",
"Sarah Ellen O'Connor",
"Sébastien Besseau",
"Vincent Courdavault",
"Clément Cuello",
"Emily Amor Stander",
"Hans J. Jansen",
"Thomas Dugé De Bernonville",
"Audrey Oudin",
"Caroline Birer Williams",
"Arnaud Lanoue",
"Nathalie Giglioli Guivarc'h",
"Nicolas Papon",
"Ron P. Dirks",
"Michael Krogh Jensen",
"Sarah Ellen O'Connor",
"Sébastien Besseau"
],
"abstract": "The Madagascar periwinkle, Catharanthus roseus, belongs to the Apocynaceae family. This medicinal plant, endemic to Madagascar, produces many important drugs including the monoterpene indole alkaloids (MIA) vincristine and vinblastine used to treat cancer worldwide. Here, we provide a new version of the C. roseus genome sequence obtained through the combination of Oxford Nanopore Technologies long-reads and Illumina short-reads. This more contiguous assembly consists of 173 scaffolds with a total length of 581.128 Mb and an N50 of 12.241 Mb. Using publicly available RNAseq data, 21,061 protein coding genes were predicted and functionally annotated. A total of 42.87% of the genome was annotated as transposable elements, most of them being long-terminal repeats. Together with the increasing access to MIA-producing plant genomes, this updated version should ease evolutionary studies leading to a better understanding of MIA biosynthetic pathway evolution.",
"keywords": [
"Monoterpene indole alkaloids",
"Catharanthus roseus",
"Apocynaceae"
],
"content": "Introduction\n\nThe Madagascar periwinkle, Catharanthus roseus (L.) G. Don, is an Apocynaceae plant native to Madagascar. C. roseus produces several specialized metabolites including monoterpene indole alkaloids (MIA; O’Connor and Maresh, 2006). These molecules are produced by plants to face biotic and abiotic pressures accounting for their wide range of bioactive properties (Dugé de Bernonville et al., 2015). Above all, MIAs produced by C. roseus are well-known for being part of the human pharmacopoeia against cancer, such as the well-known vinblastine and vincristine, and other MIA derivatives, including vinorelbine (O’Connor and Maresh, 2006).\n\nDue to its high economic importance, C. roseus has extensively been studied within the last three decades becoming the model species for MIA biosynthetic pathway studies (see Pan et al., 2016 and Kulagina et al., 2022 for extensive review). C. roseus genome was firstly sequenced in 2015 (Kellner et al., 2015). Recently, a more contiguous version (v2) was generated to ease inter-species genomic comparison (Franke et al., 2019). To date, C. roseus genome sequencing and assembly did not benefit from the development of third generation sequencing technologies that lead to more contiguous genome (Jiao and Schneeberger, 2017). Thanks to these new technologies, we present here an even more contiguous genome assembly. This updated version (v2.1) should ease inter-species studies in order to better understand the diversification of MIAs and the evolution of their biosynthetic pathways.\n\n\nMethods\n\nC. roseus cv ‘SunStorm® Apricot’ seeds (variety ID: 70001114, Syngenta flowers, Basel, Switzerland) were greenhouse-grown at the University of Tours for 1 month before sampling. DNA was extracted from C. roseus leaves using Qiagen Plant DNeasy kit (ID: 69204, Qiagen, Hilden, Germany) following the manufacturer’s instructions. Illumina sequencing library were constructed using the TruSeq DNA PCR-free kit (ID: 20015962, Illumina, San Diego, USA) and sequenced in paired-end mode (2 × 150 bp) by Eurofins Genomics (Les Ulis, France) using Illumina NextSeq500 technology. Future Genomics Technologies (Leiden, The Netherland) constructed ONT library using ONT 1D ligation sequencing kit (SQK-LSK109, Oxford Nanopore Technologies Ltd, Oxford, United-Kingdom) subsequently sequenced on Nanopore GridION flowcell and Nanopore PromethION flowcell (Oxford Nanopore Technologies Ltd, Oxford, United-Kingdom) with the GuPPy (RRID:SCR_022353) version 3.2.6 high-accuracy basecaller. A total of 114,329,683 paired-end reads were obtained from the Illumina HiSeq sequencing, 908,999 and 2,588,997 from the ONT GridION and ONT PromethION sequencing, respectively.\n\nThe C. roseus genome was assembled by Future Genomics Technologies (Leiden, The Netherlands). After adapters removal using Porechop (RRID:SCR_016967) (Wick et al., 2017), ONT reads were first assembled into contig using Flye (RRID:SCR_017016) assembler (v.2.5, Kolmogorov et al., 2019) with the following options: --min-overlap 10000 -i 2. Redundant contigs were removed using Purge_haplotigs (RRID:SCR_017616) (v.1.1.0) followed by two rounds of polishing with Illumina paired-end reads using Pilon (RRID:SCR_014731) (v.1.23, Walker et al., 2014).\n\nRNA-seq data were retrieved from the NCBI Sequence Read Archive (SRA) (RRID:SCR_004891) database using the following accession numbers: ERS1229288, ERS1229289, ERS1229290, ERS1229291, ERS1229292, ERS1229293, ERS1229294, ERS1229295, ERS1229296, ERS1907920, ERS2396963, ERS2396964, ERS2396965, ERS2396966, SRR20661631. These data were individually aligned to the C. roseus genome using HISAT2 (RRID:SCR_015530) (v.2.2.1, Kim et al., 2019). Transcripts were subsequently assembled using the resulting RNA-seq alignments and StringTie (RRID:SCR_016323) (v.2.1.7, Pertea et al., 2015). These individual transcriptomes were further merged using stringtie-merge to a non-redundant set of transcripts. A combination of similarity search using BLASTX (RRID:SCR_001653) and BLASTP (v.2.6.0-1, Camacho et al. 2009) against UniProt (RRID:SCR_002380) database (v.2022-10-12) and hmmscan (v.3.1b2, Finn et al., 2011) against the Pfam (RRID:SCR_004726) database was used to assign putative function to each gene model.\n\nThe stat program from BBmap (RRID:SCR_016965) tool (v.38.94, Bushnell, 2014) was used to assess assembly quality. Benchmarking Universal Single-Copy Orthologs (BUSCO v.5.2.2, Simão et al., 2015) (RRID:SCR_015008) with default settings was used to assess genome and gene models completeness using a plant-specific database of 2,326 single copy orthologs (eudicots_odb10). The agat_sp_statistics perl script from the AGAT package (v.0.8.0, Dainat et al., 2022) was used to get the gene models statistics.\n\nIdentification and annotation of transposable elements was determined using extensive de novo TE annotator (EDTA v.1.9.5, Ou et al., 2019) (RRID:SCR_022063) using the sensitive mode. This pipeline annotates long-terminal repeat (LTR) using LTR_Finder (RRID:SCR_015247) (v. 1.07, Xu and Wang, 2007) and LTRharvest (RRID:SCR_018970) included in GenomeTools (RRID:SCR_016120) (v.1.5.10, Ellinghaus et al., 2008); terminal inverted repeat (TIR) using Generic repeat finder (v.1.0, Shi and Liang, 2019) and TIR-learner (v.2.5, Su et al., 2019); and Helitrons using HelitronScanner (v.1.1, Xiong et al., 2014). TE size thresholds are further used to prevent false discoveries. Hence, TIR shorter than 80 bp as well as LTR and Helitrons shorter than 100 bp are considered as tandem repeats and short sequences. To prevent false LTR discoveries, LTR are further filtered using LTR_retriever (RRID:SCR_017623) (v.2.9.0, Ou and Jiang, 2018). TIR candidates are classified as MITEs if not exceeding 600 bp. TIR and Helitrons are further filtered using EDTA advanced filters (see Ou et al., 2019 for details). The genome is then masked using the obtained TE library. Unmasked part of the genome is then scanned by RepeatModeler (RRID:SCR_015027) (v.2.0.1, default parameters, Flynn et al., 2020) to identify non-LTR retrotransposons and unclassified TE missed by structure-based TE identification tools. Finally, EDTA uses the provided CDS sequences to remove gene-related sequences.\n\n\nResults\n\nC. roseus genome was assembled from ONT long-reads using Flye (v.2.5) resulting in a 651.9 Mb assembly distributed across 788 contigs. This assembly was collapsed using purge_haplotigs into 173 scaffolds reducing length to 585,8 Mb but increasing N50 from 10.3 Mb to 12.3 Mb. Assembly polishing was performed twice using Illumina short-reads with pilon (v. 1.23). C. roseus final assembly consisted in 173 scaffolds with a total length of 581.45 Mb. Even though C. roseus v.2.1 displayed similar BUSCO scores compared to C. roseus v.2 based on Eudicotyledons Benchmarking Universal Single-Copy Orthologs (BUSCO), this new version v.2.1 turns out to be much more contiguous with a 12 time less contigs and a six-fold larger N50 (Table 1) (Cuello et al., 2022).\n\na Number of scaffolds.\n\nb BUSCO scores (genome mode) % Complete [% Complete and single-copy; % Complete and Duplicated]; % Fragmented; % Missing (n = 2,326).\n\nRNA-seq based gene model prediction using publicly available data resulted in a total of 21,061 genes. Despite less genes were annotated; a higher BUSCO score was obtained (Figure 1). The combination of BLASTP and BLASTX against UniProt database and hmmscan against the PFAM database led to the functional annotation of 76.5% of the predicted genes (16,118 of the 21,062 genes, Supplementary Table S1 in Underlying data (Cuello et al., 2022)). All functionally validated MIA biosynthetic genes from C. roseus could be found in this new version v.2.1 of the genome with identity and coverage percentage ranging from 95 to 100% and 94 to 100%, respectively, with the exception of G10H and DAT (Supplementary Table S2-S3 in Underlying data (Cuello et al., 2022)).\n\nBUSCO: Benchmarking Universal Single-Copy Orthologs.\n\nFinally, we analyzed TE composition of this updated C. roseus genome. While 38.78% of the genome consisted in TE in C. roseus v.2, a higher proportion (42.87%) was annotated as TE in this new version (v.2.1) with similar distribution across the different TE families (Figure 2). It is worth noting that TE proportion of this v.2.1 is closer to the one in its recently sequenced closely related species Vinca minor (Stander et al., 2022).\n\nTIR: terminal inverted repeat, LTR: long terminal repeat, non LTR: retrotransposons without LTR sequence, other LTR: LTR containing retrotransposons except for Gypsy and Copia.",
"appendix": "Data availability\n\nBioProject: Catharanthus roseus genome sequencing. Raw sequence reads, complete genome. Accession number PRJNA907167, https://identifiers.org/NCBI/bioproject:PRJNA907167 (Tours University, 2022a).\n\nBioSample: Plant sample from Catharanthus roseus, Accession number SAMN31953452, https://identifiers.org/NCBI/biosample:SAMN31953452 (Tours University, 2022b).\n\nFigshare: An updated version of Catharanthus roseus genome. 10.6084/m9.figshare.21641111 (Cuello et al., 2022).\n\nThis project contains the following underlying data:\n\n• Catharanthus_roseus_v2.1_UT.cds (Predicted CDS).\n\n• Catharanthus_roseus_v2.1_UT.gff (Genome annotation file (GFF)).\n\n• Catharanthus_roseus_v2.1_UT.pep (Predicted proteins).\n\n• Catharanthus_roseus_v2.1_UT.tr (Predicted transcripts).\n\n• Cuello et al – F1000R – SuppMat.xlsx (Supplementary tables).\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).\n\n\nAcknowledgments\n\nThe authors benefitted from the use of the cluster at the Centre de Calcul Scientifique en région Centre-Val de Loire.\n\n\nReferences\n\nBushnell B: BBMap: A Fast, Accurate, Splice-Aware Aligner (No. LBNL-7065E). Berkeley, CA (United States):Lawrence Berkeley National Lab. (LBNL);2014.\n\nCamacho C, Coulouris G, Avagyan V, et al.: BLAST+: architecture and applications. BMC Bioinformatics. 2009; 10: 421. PubMed Abstract | Publisher Full Text | Free Full Text\n\nCuello C, Stander E, Jansen HJ, et al.:An updated version of the Madagascar periwinkle genome. figshare. [Dataset].2022. Publisher Full Text\n\nDainat J, Hereñú D; LucileSol, pascal-git: NBISweden/AGAT: AGAT-v0.8.1. Zenodo. 2022. Publisher Full Text\n\nDugé de Bernonville T, Clastre M, Besseau S, et al.: Phytochemical genomics of the Madagascar periwinkle: Unravelling the last twists of the alkaloid engine. Phytochemistry. 2015; 113: 9–23. PubMed Abstract | Publisher Full Text\n\nEllinghaus D, Kurtz S, Willhoeft U: LTRharvest, an efficient and flexible software for de novo detection of LTR retrotransposons. BMC Bioinformatics. 2008; 9(1): 18. PubMed Abstract | Publisher Full Text | Free Full Text\n\nFinn RD, Clements J, Eddy SR: HMMER web server: interactive sequence similarity searching. Nucleic Acids Res. 2011; 39: W29–W37. PubMed Abstract | Publisher Full Text | Free Full Text\n\nFlynn JM, Hubley R, Goubert C, et al.: RepeatModeler2 for automated genomic discovery of transposable element families. PNAS. 2020; 117(17): 9451–9457. PubMed Abstract | Publisher Full Text | Free Full Text\n\nFranke J, Kim J, Hamilton JP, et al.: Gene Discovery in Gelsemium Highlights Conserved Gene Clusters in Monoterpene Indole Alkaloid Biosynthesis. ChemBioChem. 2019; 20: 83–87. PubMed Abstract | Publisher Full Text\n\nJiao WB, Schneeberger K: The impact of third generation genomic technologies on plant genome assembly. Curr. Opin. Plant Biol. 2017; 36: 64–70. PubMed Abstract | Publisher Full Text\n\nKellner F, Kim J, Clavijo BJ, et al.: Genome-guided investigation of plant natural product biosynthesis. Plant J. 2015; 82: 680–692. PubMed Abstract | Publisher Full Text\n\nKim D, Paggi JM, Park C, et al.: Graph-based genome alignment and genotyping with HISAT2 and HISAT-genotype. Nat. Biotechnol. 2019; 37(8): 907–915. PubMed Abstract | Publisher Full Text | Free Full Text\n\nKolmogorov M, Yuan J, Lin Y, et al.: Assembly of long, error-prone reads using repeat graphs. Nat. Biotechnol. 2019; 37: 540–546. PubMed Abstract | Publisher Full Text\n\nKulagina N, Méteignier LV, Papon N, et al.: More than a Catharanthus plant: A multicellular and pluri-organelle alkaloid-producing factory. Curr. Opin. Plant Biol. 2022; 67: 102200. PubMed Abstract | Publisher Full Text\n\nO’Connor SE, Maresh JJ: Chemistry and biology of monoterpene indole alkaloid biosynthesis. Nat. Prod. Rep. 2006; 23: 532–547. PubMed Abstract | Publisher Full Text\n\nOu S, Jiang N: LTR_retriever: a highly accurate and sensitive program for identification of long terminal repeat retrotransposons. Plant Physiol. 2018; 176(2): 1410–1422. PubMed Abstract | Publisher Full Text | Free Full Text\n\nOu S, Su W, Liao Y, et al.: Benchmarking transposable element annotation methods for creation of a streamlined, comprehensive pipeline. Genome Biol. 2019; 20: 275. PubMed Abstract | Publisher Full Text | Free Full Text\n\nPan Q, Mustafa NR, Tang K, et al.: Monoterpenoid indole alkaloids biosynthesis and its regulation in Catharanthus roseus: a literature review from genes to metabolites. Phytochem. Rev. 2016; 15: 221–250. Publisher Full Text\n\nPertea M, Pertea GM, Antonescu CM, et al.: StringTie enables improved reconstruction of a transcriptome from RNA-seq reads. Nat. Biotechnol. 2015; 33(3): 290–295. PubMed Abstract | Publisher Full Text | Free Full Text\n\nShi J, Liang C: Generic repeat finder: a high-sensitivity tool for genome-wide de novo repeat detection. Plant Physiol. 2019; 180(4): 1803–1815. PubMed Abstract | Publisher Full Text | Free Full Text\n\nSimão FA, Waterhouse RM, Ioannidis P, et al.: BUSCO: assessing genome assembly and annotation completeness with single-copy orthologs. Bioinformatics. 2015; 31: 3210–3212. Publisher Full Text\n\nStander EA, Cuello C, Birer-Williams C, et al.: The Vinca minor genome highlights conserved evolutionary traits in monoterpene indole alkaloid synthesis. G3 Genes|Genomes|Genetics. 2022; 12: jkac268. PubMed Abstract | Publisher Full Text | Free Full Text\n\nSu W, Gu X, Peterson T: TIR-learner, a new ensemble method for TIR transposable element annotation, provides evidence for abundant new transposable elements in the maize genome. Mol. Plant. 2019; 12(3): 447–460. PubMed Abstract | Publisher Full Text\n\nTours University:Catharanthus roseus genome. [Dataset]. BioProject. 2022a.Reference Source\n\nTours University:Plant sample from Catharanthus roseus. [Dataset]. BioSample. 2022b.Reference Source\n\nWalker BJ, Abeel T, Shea T, et al.: Pilon: An Integrated Tool for Comprehensive Microbial Variant Detection and Genome Assembly Improvement. PLoS One. 2014; 9: e112963–944 e112963. PubMed Abstract | Publisher Full Text | Free Full Text\n\nWick RR, Judd LM, Gorrie CL, et al.: Completing bacterial genome assemblies with multiplex MinION sequencing. Microb. Genom. 2017; 3(10): e000132. PubMed Abstract | Publisher Full Text | Free Full Text\n\nXiong W, He L, Lai J, et al.: HelitronScanner uncovers a large overlooked cache of Helitron transposons in many plant genomes. Proc. Natl. Acad. Sci. USA. 2014; 111(28): 10263–10268. PubMed Abstract | Publisher Full Text | Free Full Text\n\nXu Z, Wang H: LTR_FINDER: an efficient tool for the prediction of full-length LTR retrotransposons. Nucleic Acids Res. 2007; 35(Web Server issue): W265–W268. PubMed Abstract | Publisher Full Text | Free Full Text"
}
|
[
{
"id": "158640",
"date": "04 Jan 2023",
"name": "Evangelos Tatsis",
"expertise": [
"Reviewer Expertise Plant specialised metabolism"
],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe current work reports an updated version of the significant medicinal plant Catharanthus roseus. C. roseus is the only source of the clinically used anticancer drug vinblastine and such work can provide resources for molecular breeding to improve the production of vinblastine and other MIAs.\nThe sequencing techniques and the bioinformatic analysis are appropriate. I recommend the current manuscript for indexing as it is.\n\nAre the rationale for sequencing the genome and the species significance clearly described? Yes\n\nAre the protocols appropriate and is the work technically sound? Yes\n\nAre sufficient details of the sequencing and extraction, software used, and materials provided to allow replication by others? Yes\n\nAre the datasets clearly presented in a usable and accessible format, and the assembly and annotation available in an appropriate subject-specific repository? Yes",
"responses": []
},
{
"id": "158636",
"date": "06 Feb 2023",
"name": "Amit Rai",
"expertise": [
"Reviewer Expertise Genome sciences",
"Metabolomics research. Evolutionary biology",
"MIA biosynthesis pathways"
],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 presented article, authors reported an updated version of genome assembly for Madagascar periwinkle, which is a valuable model plant species to study MIA biosynthesis. Compared to the previously published genome assemblies for Madagascar periwinkle, this study used long read sequencing technology and achieved an improvement in terms of contig N50.\nWithout a doubt, this is a better genome assembly, but authors should have considered scaffolding through HiC to achieve a chromosome-scale genome assembly as that would have allowed them to discover novel features contributing MIA biosynthesis and evolution.\nNevertheless, the resource presented here is valuable, and will inspire researchers to combine the generated datasets in this study with new sequencing data to derive a chromosome-scale genome assembly for C. roseus. For these reasons, I support its indexing.\n\nAre the rationale for sequencing the genome and the species significance clearly described? Yes\n\nAre the protocols appropriate and is the work technically sound? Yes\n\nAre sufficient details of the sequencing and extraction, software used, and materials provided to allow replication by others? Yes\n\nAre the datasets clearly presented in a usable and accessible format, and the assembly and annotation available in an appropriate subject-specific repository? Yes",
"responses": []
}
] | 1
|
https://f1000research.com/articles/11-1541
|
https://f1000research.com/articles/11-1538/v1
|
20 Dec 22
|
{
"type": "Method Article",
"title": "BOREALIS: an R/Bioconductor package to detect outlier methylation from bisulfite sequencing data",
"authors": [
"Gavin R. Oliver",
"W. Garrett Jenkinson",
"Rory J. Olson",
"Laura E. Schultz-Rogers",
"Eric W. Klee",
"Gavin R. Oliver",
"W. Garrett Jenkinson",
"Rory J. Olson",
"Laura E. Schultz-Rogers"
],
"abstract": "Background: Rare genetic disease studies have benefited from the era of high throughput sequencing. DNA sequencing results in genetic diagnosis of 18-40% of previously unsolved cases, while the incorporation of RNA-Seq analysis has more recently been shown to generate significant numbers of previously unattainable diagnoses. While DNA methylation remains less explored, multiple inborn diseases resulting from disorders of genomic imprinting are well characterized and a growing body of literature suggests the causative or correlative role of aberrant methylation in diverse rare inherited conditions. Complex pictures of methylation patterning are also emerging, including the association of regional, multiple specific-site or even single-site methylation, with disease. The systematic application of genomic-wide methylation-based sequencing for undiagnosed cases of rare diseases is a logical progression from current testing paradigms. Similar to the rationale previously exploited in RNA-based rare disease studies, we can assume that disease-associated or causative methylation aberrations in an individual will demonstrate significant differences from other individuals with unrelated phenotypes. Thus, aberrantly methylated sites will be outliers from a heterogeneous cohort of individuals. Methods: Based on this rationale, we present BOREALIS: Bisulfite-seq OutlieR MEthylation At SingLeSIte ReSolution. BOREALIS uses a beta binomial model to identify outlier methylation at single CpG site resolution from bisulfite sequencing data. Results: Utilizing power analyses, we demonstrate that BOREALIS can identify outlier CpG methylation within a cohort of samples. Furthermore, we show that BOREALIS is tolerant to the inclusion of multiple identical outliers with sufficient cohort size and sequencing depth. Conclusions: The method demonstrates improved performance versus standard statistical testing and is suited for single or multi-site downstream analysis.",
"keywords": [
"methylation",
"outlier",
"rare disease",
"diagnostic odyssey"
],
"content": "Introduction\n\nMultiple inborn diseases resulting from disorders of genomic methylation are well characterized. A growing body of literature reports associations between DNA methylation and conditions including Parkinson’s Disease (Chuang, et al., 2017) and methylation-based studies have also suggested the causative or correlative role of aberrant methylation in diverse rare inherited conditions (Guastafierro, et al., 2017; Sharp, et al., 2017; Sobreira, et al., 2017).\n\nMethods for profiling DNA methylation traditionally focused on wide genomic regions, particularly CpG islands. While microarray methylation profiling continues to be utilized, next-generation sequencing has enabled analysis at read count-level resolution and greatly increased numbers of genomic CpG sites (Wu, et al., 2015). High-resolution methodologies have enabled complex pictures of methylation to emerge, including association of multiple specific-site or even single-site methylation with developmental processes or disease (Bui, et al., 2012; Choi, et al., 2018; Claus, et al., 2012; Fürst, et al., 2012; Hashimoto, et al., 2013; Nile, et al., 2008; Pogribny, et al., 2000; Scantamburlo, et al., 2017; Sohn, et al., 2010; Takahashi, et al., 2017).\n\nDNA sequencing results in the diagnosis of up to 40% of genetic disease cases previously unsolved using standard clinical testing (Sawyer, et al., 2016). RNA-Seq has been investigated and has shown benefit in complementing DNA testing (Cummings, et al., 2017). With the growing evidence linking methylation to disease, the application of methylation-based sequencing for undiagnosed cases of rare inherited disease is a logical progression from current testing paradigms. Expanded methylation profiling will offer the ability to detect diagnostic signals unique to the epigenome, undetectable in DNA and RNA due to lack of measurable manifestation in those materials, or due to shortcomings in current technologies or analytical approaches.\n\nAn ideal method to detect deviant methylation should offer the ability to profile at single CpG sites while enabling flexibility to consolidate calls across regions. In the context of genetic disease, we can assume that disease-associated methylation aberrations in an individual will show significant differences from individuals with unrelated phenotypes. A similar rationale was successfully used by us and others in outlier-based RNA analysis (Jenkinson, et al., 2020). However, existing solutions for the detection of differentially methylated CpG sites from bisulfite sequencing focus on traditional group vs group or multi-group experimental designs (Wreczycka, et al., 2017) and are therefore not suited to genetic disease or other outlier-based analyses (Figure 1).\n\nWhile traditional approaches to differential methylation analysis are based on group case vs. control analysis, BOREALIS utilizes a one vs. many outlier approach whereby individual(s) are compared to a cohort. This approach is especially useful when multiple similar cases are difficult to identify, as is the case in rare genetic disease studies. By comparing every affected individual to a cohort of heterogeneous individuals, outlier methylation can be identified at individual CpG sites for all members of the cohort, without the requirement for multiple similar cases.\n\n\nMethods\n\nAt a given CpG site, we assume we have data in the form of methylated counts xi and total read counts ni for individuals i = 1, …, I in a cohort of size I. If every individual in the population had the exact same probability p of methylation at this site, i.e., p1 = p2 = … = pi = p where pi is the (true-but-unknown) probability of methylation for the ith individual at this site, then the methylated counts xi would be binomially distributed with parameters p and ni. However, we expect varying degrees of sample-to-sample variability in the probability of methylation at a given site even in a healthy cohort. Therefore, it is more biologically accurate to assume that pi for i=1, …, I have been sampled from a distribution over the unit interval. A common and mathematically convenient choice for this distribution is a beta distribution with parameters α and β. Thus the observed number of methylated reads xi for the ith individual can be viewed as being generated from a two-step process whereby the probability of methylation is selected pi ~ Beta (α,β) and given this probability the number of methylated reads is binomially distributed xi|pi ~ Binomial (pi,ni). Viewed in this way, we say the methylated reads are beta-binomially distributed xi ~ Beta-Binomial (ni,α,β) with parameters α and β, and popular packages for differential DNA-methylation detection such as Dispersion Shrinkage for Sequencing (DSS) (Feng and Wu, 2019) use this same distribution for methylated counts.\n\nWhat BOREALIS does differently from traditional tools such as DSS (Park and Wu, 2016) is that it builds its statistical model explicitly for the purpose of outlier detection compared to a cohort, which requires alternative statistical framing and considerations as compared to group-versus-group analyses (Jenkinson, et al., 2020). Specifically, at each CpG site, BOREALIS takes the input data {(xi,ni): i = 1, …, I} and estimates the population-level parameters α and β using gamlss library (https://www.gamlss.com) (Feng and Wu, 2019). We implement Laplace Smoothing on the counts (i.e. we use as counts xi~= xi+1 and ni~= ni+2) as a regularization step to help deal with any samples with low counts. From these estimated α and β parameters, we (for the ith sample) compute the left-sided p-value by looking at the probability that a value of xi or fewer methylated reads were generated from a Beta-Binomial (ni,α,β), and likewise a right-sided p-value would evaluate the probability that a value of xi or greater methylated reads came from this distribution. We implement this probability calculation using the pBB function of gamlss. The two-sided p-value is computed as two times the lesser of these one-sided p-values.\n\nTo validate performance of the BOREALIS method, we performed Monte Carlo simulations of an outlier sample in cohorts of varying sizes sequenced at varying depths of coverage. Namely, after selecting a cohort size I and an average depth of coverage D we conducted 10,000 Monte Carlo simulations wherein each sample i in the cohort has sequencing depth di drawn from a Poisson distribution with mean D. The number of methylated reads in cohort sample i is drawn from a Beta-Binomial distribution with mean 0.8 and dispersion 0.1, and then these simulated cohort data are fit using the BOREALIS model. An outlier sample is then simulated with sequencing depth d drawn from a Poisson distribution with mean D and number of methylated reads given by a Binomial distribution with mean 0.3. BOREALIS is then used to compute a p-value thresholded at level 0.05, and the power is given by the fraction of the 10,000 simulations that correctly reject the null hypothesis. Full code to replicate the power analysis is provided as described in the Data Availability section.\n\n\nResults\n\nThe results of the power analysis are plotted in Figure 2 demonstrating that BOREALIS can accurately detect outlier methylation events in modest cohort sizes and sequencing depths. BOREALIS can successfully identify outlier methylation with high statistical power utilizing a wide range of sample numbers (3-100+) and read depths (< 10 – 100s). BOREALIS is also tolerant of multiple identical outliers provided sufficient sequencing depth and cohort size as shown in Figure 3. The method supports multithreaded computation, as well as splitting across chromosomes to facilitate parallelism across compute nodes in a cluster environment. To provide users with the ability to visually review the methylation distributions underlying any call made by BOREALIS, we provide a built-in plotting function whose outputs are illustrated in Figure 4.\n\nGraphical summarization of Monte Carlo simulations of an outlier sample in a cohort of varying sizes and depths of sequencing coverage. Ten thousand simulations were performed for each set of experimental conditions whereby random sampling of sequencing depth for each sample and the proportion methylated reads was performed. BOREALIS built its beta-binomial model for each simulated cohort and an outlier sample was simulated with BOREALIS used to compute a p-value. Power estimation is based on the simulations correctly rejecting the null hypothesis at a level p < = 0.05. The parameters mu (mean methylation fraction at a given site), sigma (variability in methylation fraction at a given site) and muAb (deviation from the mean methylation level in the outlier sample) are fixed for the purposes of the simulation shown.\n\nGraphical summarization of Monte Carlo simulations of (A) two outlier samples and (B) three outlier samples in a cohort of varying sizes and depths of sequencing coverage. While rare disease studies generally aim to identify single outlier individuals in a cohort, this demonstrates the ability of the BOREALIS approach to identify multiple, identical outliers in the presence of sufficient read coverage and cohort size. One thousand simulations were performed for each set of experimental conditions whereby random sampling of sequencing depth for each sample and the proportion methylated reads\n\nwas performed. BOREALIS built its beta-binomial model for each simulated cohort and an outlier sample was simulated with BOREALIS used to compute a p-value.\n\nHere a single site within the LTB4R gene promoter is shown for Patient 72, from the BOREALIS Bioconductor package’s included test data. Full details on how to perform BOREALIS analysis and generate similar figures are detailed in the BOREALIS vignette, included with the package.\n\nBOREALIS is packaged with a vignette that will enable new users to become quickly familiar with program outputs and potential downstream use-cases. These include topics including:\n\n1) Running the core BOREALIS method on a cohort\n\n2) Post-processing\n\n3) Generating summary metrics\n\n4) Annotating program outputs with user-defined genomic features\n\n5) Generating visual outputs for single-site data\n\n6) Summarizing single-site data across genomic features\n\nThe vignette is available in HTML format within Bioconductor online (https://bioconductor.org/packages/release/bioc/vignettes/borealis/inst/doc/borealis.html) or packaged with the Bioconductor package itself. This vignette provides a hands-on introduction to the package and will be beneficial for new users, prior to them developing their own specific workflows.\n\n\nConclusions\n\nBOREALIS is a novel R/Bioconductor package that addresses an unmet need in bisulfite sequencing-based genetic disease studies. The method is suited for single or multi-site downstream analysis and can successfully identify outlier methylation with high statistical power, providing a new avenue of exploration in the quest for increased diagnostic rates in genetic disease patients. It is readily available and easily implemented, enabling seamless integration with other common pipelines and tools.\n\n\nAuthor contributions\n\nGRO’s contributions include Data Curation, Formal Analysis, Investigation, Methodology, Software, Supervision, Validation, Visualization, Writing – Original Draft Preparation, Writing – Review & Editing. WGJ was involved with Conceptualization, Data Curation, Formal Analysis, Methodology, Project Administration, Software, Supervision, Validation, Visualization, Writing – Review & Editing. RJO and LESR participated in study design, helped with analysis and edited the manuscript. EWK participated in study design, secured funding and edited the manuscript.\n\n\nSoftware availability\n\n\n\n• Software available from: https://bioconductor.org/packages/release/bioc/html/borealis.html\n\n• Source code available from: https://github.com/GarrettJenkinson/borealis\n\n• Archived source code at time of publication: Zenodo (DOI: https://doi.org/10.5281/zenodo.7342710) (Oliver, et al., 2022a)\n\n• License: GNU General Public License v3.0. (GPL -3)",
"appendix": "Data availability\n\nZenodo. BOREALIS Power Analysis Code and Data. (DOI: https://doi.org/10.5281/zenodo.7343136) (Oliver, et al., 2022b)\n\nThe project contains the following underlying data:\n\n• Fig 2.csv (Power analysis data for Figure 2 from the manuscript)\n\n• Fig 2.pdf (Figure 2 power analysis graph in PDF format)\n\n• Fig 2_power_analysis.R (R code to regenerate the power analysis and csv output for Figure 2)\n\n• Fig 3A.csv (Power analysis data for Figure 3A from the manuscript)\n\n• Fig 3A.pdf (Figure 3A power analysis graph in PDF format)\n\n• Fig 3A_power_analysis.R (R code to regenerate the power analysis and csv output for Figure 3A)\n\n• Fig 3B.csv (Power analysis data for Figure 3B from the manuscript)\n\n• Fig 3B.pdf (Figure 3B power analysis graph in PDF format)\n\n• Fig 3B_power_analysis.R (R code to regenerate the power analysis and csv output for Figure 3B)\n\n• README.txt (Text file with instructions to regenerate power analysis data and graphs)\n\n• plotFig2.R (R code to generate the graph for Figure 2 using the csv input)\n\n• plotFig3.R (R code to generate the graph for Figure 3A and 3B using the csv input)\n\nData is under a Creative Commons Attribution 4.0 International license.\n\n\nAcknowledgements\n\nAn earlier version of this article can be found in “Detection of outlier methylation from bisulfite sequencing data with novel Bioconductor package BOREALIS” (doi: https://doi.org/10.1101/2022.05.19.492700).\n\n\nReferences\n\nBui C, et al.: cAMP response element-binding (CREB) recruitment following a specific CpG demethylation leads to the elevated expression of the matrix metalloproteinase 13 in human articular chondrocytes and osteoarthritis. FASEB J. 2012; 26(7): 3000–3011. PubMed Abstract | Publisher Full Text\n\nChoi NY, et al.: Novel imprinted single CpG sites found by global DNA methylation analysis in human parthenogenetic induced pluripotent stem cells. Epigenetics. 2018; 13(4): 343–351. PubMed Abstract | Publisher Full Text | Free Full Text\n\nChuang Y-H, et al.: Parkinson’s disease is associated with DNA methylation levels in human blood and saliva. Genome Med. 2017; 9(1): 76. PubMed Abstract | Publisher Full Text | Free Full Text\n\nClaus R, et al.: Quantitative DNA Methylation Analysis Identifies a Single CpG Dinucleotide Important for ZAP-70 Expression and Predictive of Prognosis in Chronic Lymphocytic Leukemia. J. Clin. Oncol. 2012; 30(20): 2483–2491. PubMed Abstract | Publisher Full Text | Free Full Text\n\nCummings BB, et al.: Improving genetic diagnosis in Mendelian disease with transcriptome sequencing. Sci. Transl. Med. 2017; 9(386). PubMed Abstract | Publisher Full Text | Free Full Text\n\nFeng H, Wu H: Differential methylation analysis for bisulfite sequencing using DSS. Quant Biol. 2019; 7(4): 327–334. PubMed Abstract | Publisher Full Text | Free Full Text\n\nFürst RW, et al.: A differentially methylated single CpG-site is correlated with estrogen receptor alpha transcription. J. Steroid Biochem. Mol. Biol. 2012; 130(1-2): 96–104. PubMed Abstract | Publisher Full Text\n\nGuastafierro T, et al.: Genome-wide DNA methylation analysis in blood cells from patients with Werner syndrome. Clin. Epigenetics. 2017; 9(1): 92. PubMed Abstract | Publisher Full Text | Free Full Text\n\nHashimoto K, et al.: Regulated transcription of human matrix metalloproteinase 13 (MMP13) and interleukin-1β (IL1B) genes in chondrocytes depends on methylation of specific proximal promoter CpG sites. J. Biol. Chem. 2013; 288(14): 10061–10072. PubMed Abstract | Publisher Full Text | Free Full Text\n\nJenkinson G, et al.: LeafCutterMD: an algorithm for outlier splicing detection in rare diseases. Bioinformatics. 2020; 36(17): 4609–4615. PubMed Abstract | Publisher Full Text | Free Full Text\n\nNile CJ, et al.: Methylation status of a single CpG site in the IL6 promoter is related to IL6 messenger RNA levels and rheumatoid arthritis. Arthritis & Rheumatism. 2008; 58(9): 2686–2693. PubMed Abstract | Publisher Full Text\n\nOliver GR, Jenkinson WG, Klee EW:BOREALIS: an R/Bioconductor package to detect outlier methylation from bisulfite sequencing data (3.15).Zenodo.2022a. Publisher Full Text\n\nOliver GR, Jenkinson WG, Klee EW:BOREALIS Power Analysis Code and Data (1.0). [Data set]. Zenodo.2022b. Publisher Full Text\n\nPark Y, Wu H: Differential methylation analysis for BS-seq data under general experimental design. Bioinformatics. 2016; 32(10): 1446–1453. PubMed Abstract | Publisher Full Text\n\nPogribny IP, et al.: Single-site methylation within the p53 promoter region reduces gene expression in a reporter gene construct: possible in vivo relevance during tumorigenesis. Cancer Res. 2000; 60(3): 588–594. PubMed Abstract\n\nSawyer SL, et al.: Utility of whole-exome sequencing for those near the end of the diagnostic odyssey: time to address gaps in care. Clin. Genet. 2016; 89(3): 275–284. PubMed Abstract | Publisher Full Text | Free Full Text\n\nScantamburlo G, et al.: Interleukin-4 Induces CpG Site-Specific Demethylation of the Pendrin Promoter in Primary Human Bronchial Epithelial Cells. Cell. Physiol. Biochem. 2017; 41(4): 1491–1502.\n\nSharp GC, et al.: Distinct DNA methylation profiles in subtypes of orofacial cleft. Clin. Epigenetics. 2017; 9(1): 63. PubMed Abstract | Publisher Full Text | Free Full Text\n\nSobreira N, et al.: Patients with a Kabuki syndrome phenotype demonstrate DNA methylation abnormalities. Eur. J. Hum. Genet. 2017; 25(12): 1335–1344. PubMed Abstract | Publisher Full Text | Free Full Text\n\nSohn BH, et al.: Functional switching of TGF-beta1 signaling in liver cancer via epigenetic modulation of a single CpG site in TTP promoter. Gastroenterology. 2010; 138(5): 1898–1908.e12. PubMed Abstract | Publisher Full Text\n\nTakahashi A, et al.: DNA methylation of the RUNX2 P1 promoter mediates MMP13 transcription in chondrocytes. Sci. Rep. 2017; 7(1): 7771. PubMed Abstract | Publisher Full Text | Free Full Text\n\nWreczycka K, et al.: Strategies for analyzing bisulfite sequencing data. J. Biotechnol. 2017; 261: 105–115. PubMed Abstract | Publisher Full Text\n\nWu H, et al.: Detection of differentially methylated regions from whole-genome bisulfite sequencing data without replicates. Nucleic Acids Res. 2015; 43(21): e141–e141. PubMed Abstract | Publisher Full Text"
}
|
[
{
"id": "189477",
"date": "23 Aug 2023",
"name": "Philipp Jurmeister",
"expertise": [
"Reviewer Expertise DNA methylation",
"machine learning",
"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\nOliver et al. present a novel method for the detection of differentially methylated CpG sites in bisulfite sequencing read count data, together with an implementation thereof in the R software package 'borealis'. Its statistical model is specifically designed to detect a single outlier sample in a cohort of samples with consistent methylation whereas existing methods for the detection of differential methylation are designed for a group-vs-group approach. The proposed method is an adaption of the recently published LeafCutterMD package which applied the same approach to outlier splicing detection. The outlier detection setting for differential methylation does indeed seem of great relevance, specifically in the use case of the detection of rare diseases which the authors also mention. The performance of the proposed method in the shown analyses of true positive rate across sequencing depths and cohort sizes in a synthetic dataset is in itself convincing. The software package is well documented with detailed function descriptions and an extensive vignette. However, there are several weaknesses which should be addressed to improve the overall quality of the manuscript:\nMajor concerns\nThe statistical model used here does indeed directly address the described outlier detection setting and its suitability for this task seems plausible. However, we suspect existing software packages implementing a group-vs-group approach could also be applied in this setting (with group sizes 1 and l-1). We would deem a comparison of the performance in the outlier detection setting between borealis and existing software packages necessary to support the claim made about the specific suitability of borealis.\n\n(a) While the borealis R packages, as obtained from github, worked flawlessly for us, we were not able to run the code for the reproduction of Figure 2. We adjusted ‘Fig2_power_analysis.R’ to only test aveDepth <- c(80, 100), cohSize <- c(90, 100) and with nSamps = 1000. Running the script consistently yielded power = 0 for all parameters. Further inspection revealed an error occurred in line 32: ‘Error in `[.data.frame`(DatA, , ResCha) : undefined columns selected’ that was caught in exception handling. The scripts for Fig.3 and 4 showed similar behavior. We used gamlss version 5.4-12 and gamlss.dist version 6.0-5. We are happy to provide more information (e.g. full R session info) if that is considered helpful or to receive suggestions regarding potential mistakes in our handling of the code.\n(b) According to the general description of the proposed method in the methods section (and its implementation in the borealis package), the betabinomial distribution parameters for a specific CpG site are estimated on all samples of the cohort and reused for the computation of all p-values. In particular, this means that the sample to be tested is also included in the estimation of the model parameters. However, in the experiments for power analysis, the parameters are estimated not using the outlier. We feel the two approaches should be aligned.\n\nWhile the results showing the true positive rate for the detection of synthetic outlier detection are convincing, information on the false positive rate should also be provided.\nMinor concerns\nSince the proposed method contains a novel (at least for the use in methylation analysis) hypothesis test, we find that a slightly more elaborate presentation including naming hypothesis and the (admittedly simple) test statistic and its distribution would provide more clarity. An additional remark on the choice to include the tested outlier in the normal cohort for parameter estimation could also be helpful.\n\nWe found it not immediately obvious how the multiple outliers are used in the procedure to generate Figure 3 (they have identical methylation data; only one test is performed; the remaining outliers are part of the cohort that is used to estimate distribution parameters), especially since the exclusion of the tested outlier while estimating distribution parameters is not consistent with the first description of the method in the paper (see Major concern 2b). A brief description of this analysis in the methods would be helpful for an easier understanding the experiment.\n\nDSS is named as an example for a well-known R package that uses a group-vs-group approach for detecting differential methylation. We feel that more support is needed still for the claim made that all existing software packages follow this approach and borealis now fills this gap (e.g. referencing several other well-known software packages for differential methylation detection and explicitly stating their use of a group-vs-group approach), especially since this point seems central to the paper.\n\nThe introduction includes the sentence “An ideal method to detect deviant methylation should offer the ability to profile at single CpG sites while enabling flexibility to consolidate calls across regions.”. However, our understanding is that detection of differentially methylated regions is not addressed in this paper. If DMR detection can indeed be achieved with borealis, an explanation in the paper would help to highlight this. Otherwise however, we think that the functionality of borealis is sufficient as is and a deletion or rephrasing of the sentence would resolve the issue.\n\nThe performance of the proposed method has only been evaluated on a synthetic dataset. We think an experiment on real-world data would provide important data for judging the performance of the method in the clinical research use-cases mentioned in the paper.\n\nWhen evaluating the results of the borealis workflow, p-value adjustment is most likely necessary. A comment on this fact or potentially even an inclusion in borealis functionality would prevent the misconception that the p-values can be intrepreted as is.\n\nThe results subsection 'BOREALIS package vignette outline' is quite long and its content does not appear suitable for a method article to us, even though the vignette itself is very informative. Maybe briefly mentioning its existence and purpose would be more adequate?\nQuestions/Interests\nAdding to major concern 1), we would find potential theoretical insights into the properties of the group-vs-group approaches when reduced to group sizes 1 and (l-1) and a comparison to the proposed approach on a theoretical level extremely interesting.\n\nWhile the currently provided functions of the borealis package are very easy to use, several post-processing steps that are not implemented yet are still necessary for likely standard use-cases (see section 4 of the vignette). Some of these steps are not straightforward (programming-wise), others could represent potential pitfalls (pvalue correction, also see minor concern 3). We feel user-friendliness would greatly benefit from also providing functions in the borealis package for these steps, so that a full data import to interpretation pipeline is available for non-programming-savvy users.\n\nThe performance of the proposed method was evaluated for different cohort sizes and average sequencing depths. We would find it very interesting to see how varying the difference between methylation probability of the outlier and in the normal cohort affects the results.\n\nIs the rationale for developing the new method (or application) clearly explained? Partly\n\nIs the description of the method technically sound? Partly\n\nAre sufficient details provided to allow replication of the method development and its use by others? Yes\n\nIf any results are presented, are all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions about the method and its performance adequately supported by the findings presented in the article? Partly",
"responses": []
},
{
"id": "213346",
"date": "24 Oct 2023",
"name": "Vicente Yepez",
"expertise": [
"Reviewer Expertise RNA-seq based diagnostics of rare diseases",
"bioinformatics",
"statistical 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\nOliver et al., developed an R package to detect outlier methylation from bisulfite sequencing data. They use a beta binomial model for this. This can be applied in rare disease diagnostics.\nI think the method is novel and sound, but I have some concerns:\nMajor:\nIt is unclear to me what is the difference between this article and the one described in: https://www.biorxiv.org/content/10.1101/2022.05.19.492700v1.\n\nIt is unclear where the data came from.\n\nIt is unclear if no other method to detect outlier methylation from bisulfite sequencing data exists or this is the first study doing that.\n\nIt should be described how CpG sites are assigned to genes.\n\nThe Results section should describe the following:\n- the cohort including number of samples and whether they were affected. Details should be included in Methods.\n- how the methylation data was counted and # of features detected. Details should be included in Methods.\n- what were the results after applying BOREALIS to the data. How many outliers/sample were obtained.\n\n- A quantile-quantile plot of the p-values must be included to verity that they are calibrated.\n- Is there any multiple testing performed? It should, as I guess there are thousands of sites tested.\nMinor:\nIt could be stated in the Abstract the minimal number of the \"sufficient cohort size\".\n\nTo strengthen it, consider adding another citation to the sentence: \"DNA sequencing results in the diagnosis of up to 40% of genetic disease cases previously unsolved using standard clinical testing (Sawyer, et al., 2016)\".\n\nIt should be described how exactly was RNA-seq investigated in the sentence: \"RNA-Seq has been investigated and has shown benefit in complementing DNA testing (Cummings, et al., 2017)\". Also, consider adding other citations from other studies to it.\n\nOUTRIDER by Brechtmann et al., AJHG 20181, should be cited here as it was presumably the 1st method for outliers in RNA-seq data: \"A similar rationale was successfully used by us and others in outlier-based RNA analysis (Jenkinson, et al., 2020)\".\n\nOther parameters should be tested in the MC simulations besides mean 0.8 and dispersion 0.1.\n\nIs the rationale for developing the new method (or application) clearly explained? Yes\n\nIs the description of the method technically sound? Yes\n\nAre sufficient details provided to allow replication of the method development and its use by others? Yes\n\nIf any results are presented, are all the source data underlying the results available to ensure full reproducibility? No\n\nAre the conclusions about the method and its performance adequately supported by the findings presented in the article? Partly",
"responses": []
}
] | 1
|
https://f1000research.com/articles/11-1538
|
https://f1000research.com/articles/11-663/v1
|
16 Jun 22
|
{
"type": "Research Article",
"title": "Insulin and insulin-like growth factor-I receptors in astrocytes exert different effects on behavior and Alzheimer´s-like pathology",
"authors": [
"Jonathan Zegarra-Valdivia",
"Ana M. Fernandez",
"Laura Martinez-Rachadell",
"Raquel Herrero-Labrador",
"Jansen Fernandes",
"Ignacio Torres Aleman",
"Jonathan Zegarra-Valdivia",
"Ana M. Fernandez",
"Laura Martinez-Rachadell",
"Raquel Herrero-Labrador",
"Jansen Fernandes"
],
"abstract": "Background: Pleiotropic actions of insulin and insulin-like growth factor I (IGF-I) in the brain are context- and cell-dependent, but whether this holds for their receptors (insulin receptor (IR) and IGF-I receptor (IGF-IR), respectively), is less clear. Methods: We compared mice lacking IR or IGF-IR in glial fibrillary astrocytic protein (GFAP)-expressing astrocytes in a tamoxifen-regulated manner, to clarify their role in this type of glial cells, as the majority of data of their actions in brain have been obtained in neurons. Results: We observed that mice lacking IR in GFAP astrocytes (GFAP IR KO mice) develop mood disturbances and maintained intact cognition, while at the same time show greater pathology when cross-bred with APP/PS1 mice, a model of familial Alzheimer´s disease (AD). Conversely, mice lacking IGF-IR in GFAP astrocytes (GFAP-IGF-IR KO mice) show cognitive disturbances, maintained mood tone, and show control-dependent changes in AD-like pathology. Conclusions: These observations confirm that the role of IR and IGF-IR in the brain is cell-specific and context-dependent.",
"keywords": [
"Astrocytes",
"Alzheimer`s disease",
"Insulin",
"Insulin-like growth factor I",
"Glial cells."
],
"content": "Introduction\n\nWork in invertebrate insulin-like peptide (ILP) receptors, that recognize multiple ILP ligands (Kimura et al., 1997), have provided valuable information on their pleiotropy (Fernandes de Abreu et al., 2014). However, the vertebrate tyrosine kinase ILP receptors (IR) and IGF-IR specifically recognize insulin and IGF-I, respectively (McGaugh et al., 2015), making it difficult to infer their role from observations gathered in invertebrate models. For instance, daf-2, the worm ILP receptor (Kimura et al., 1997), interferes with mechanisms of proteostasis (Cohen et al., 2006) and longevity (Kenyon et al., 1993), whereas in vertebrates these roles has been tentatively assigned to IGF-IR (Cohen et al., 2009) since the role of IR in these contexts is not yet clear (Freude et al., 2009a; Shimizu et al., 2011). Moreover, the numerous actions of ILPs in physiology and pathology are context- and cell-dependent, which means that observations of the actions of ILPs in a given tissue or organ must be nuanced by the experimental approach used in each case. In brain studies, most of the information gathered on the role of IR and IGF-IR has been obtained after manipulating its function either in neurons (Gontier et al., 2015; De Magalhaes Filho et al., 2016) or in many brain cell types at the same time (Cohen et al., 2009; Soto et al., 2019).\n\nSince recently published work shows that IR and IGF-IR in astrocytes play cell-dependent actions (Cai et al., 2018; Noriega-Prieto et al., 2021), hinting to differential roles of these receptors in astrocytes, we compared behavioral traits in mice lacking IR in astrocytes with mice lacking IGF-IR in this type of cells. Mice with reduced IR in glial fibrillary astrocytic protein (GFAP) astrocytes (GFAP IR KO mice) show gradual mood disturbances and preserved cognition while mice with reduced IGF-IR in GFAP astrocytes (GFAP IGF-IR KO mice) show preserved mood and altered cognition. We also bred these mice in an APP/PS1 background mimicking familial AD-like amyloidosis and observed that GFAP IR KO mice develop significantly greater pathology whereas GFAP IGF-IR KO mice did not.\n\n\nMethods\n\nExperimental models used in this study aimed to mimic human physio-pathology in relation to the established brain insulin and IGF-I resistance during healthy aging or AD. No protocol of these studies was prepared in advance.\n\nMice were used according to Animal Research: Reporting of in vivo Experiments (ARRIVE) and this study is reported in line with the guidelines (Zegarra-Valdivia et al., 2022). Transgenic mice with tamoxifen-regulated deletion of IGF-IR or IR in astrocytes (GFAP-IGF-IR KO and GFAP-IR KO mice, respectively) were obtained as described (Garcia-Caceres et al., 2016; Noriega-Prieto et al., 2021) crossing IRf/f (B6.129S4(FVB)-Insrtm1Khn/J RRID:IMSR, Jackson labs; stock number 006955) or IGF-IRf/f (B6, 129 background; Jackson Labs; stock number: 012251) with hGFAP-CreERT2 mice (C57B&/6xSJL/J mix background Jackson Labs, stock number: 012849). To knock down the target gene, tamoxifen was administered to 2- months old mice for 5 days (75 mg/kg, Sigma, intraperitoneally) as described (Hirrlinger et al., 2006), and animals were used one month later. Controls littermates received the vehicle (corn oil). GFAP-IGF-IR KO and GFAP-IR KO display reduced mRNA levels in brain, as reported by Noriega-Prieto et al. (2021) and Garcia-Caceres et al. (2016). APPswe and PS1Δ9 mice of C57BL6/J background were from the colony of the Cajal Institute. Homozygous APP/PS1 mice were crossed with homozygous GFAP IGF-IR KO or GFAP IR KO mice to obtain the respective compound strains. Studies were carried out at the age of 10-11 months-old, when pathology is well developed.\n\nMice were were housed in standard cages (48 × 26 cm2) with 5 mice per cage. Mice were maintained on a light-dark cycle (12-12 h, lights on at 8 am) at constant temperature (22°C) and humidity, and with food (pellet rodent diet) and water ad libitum. All experimental protocols were performed during the light cycle and followed European guidelines (86/609/EEC & 2003/65/EC, European Council Directives).\n\nStudies were approved by the respective local Bioethics Committees (Government of the Community of Madrid, MERGEFIELD CÓDIGO PROEX 193.4/20 (2020) and UPV M20_2021_168 (2021). Animals were not randomized and were used in a sex-balanced manner throughout. Potential confounders were not accounted for. Each experimenter took account of group allocation under study. All efforts were done to reduce harm to the animals. Mice were handled for three days prior to any experimental manipulations and familiarized with behavioral arenas to minimize novelty stress or deeply anesthesized with pentobarbital prior to sacrifice, when needed. Sample sizes were kept as little as possible to comply with current animal reduction policies. No adverse events were expected, nor found. End-point measures included checking reflexes in deeply anesthesized animals prior to culling.\n\nThese tests were used to determine behavior under laboratory-controlled conditions. These are observational studies with no a priori hypothesis.\n\nBarnes maze. To assess spatial learning and memory, animals received reinforcement to escape from an open circular platform (92 cm Ø with 20 holes of 5 cm Ø) to the “escape chamber”, as described (Ortiz et al., 2010; Zegarra-Valdivia et al., 2019). All animals received appropriate training (four trials per day), and trials were separated by 15 min. After each trial, the maze was cleaned with 70% alcohol. On the 5th day, both groups were tested, and once more 48 hours later, evaluating the long-term memory of the animals. Time to escape to the safe chamber was quantified.\n\nOpen field. Exploratory behavior and locomotion were assessed by introducing the animal to an open field arena (42 cm× 42 cm × 30 cm, Versamax; AccuScan Instruments, Inc.) for 10 min. All parameters were quantified as described (Zegarra-Valdivia et al., 2019). Time spent exploring specific areas of the arena was measured.\n\nElevated plus maze. To assess anxiety-like/coping behavior, mice were introduced in a maze of 40 cm from the floor with two opposing arms. Two protected (closed) arms (30 cm (length) × 5 cm (wide) × 15.25 (height), and two opposing unprotected (open) arms (30 cm (length) × 5 cm (wide). Each animal was introduced in the middle of the apparatus for 5 minutes. Stress was scored as time spent in the closed arms while coping behavior was estimated by time spent in the open arms. All measures were recorded (Video Tracking Plus Maze Mouse; Med Associates, USA), and analyzed as described (Munive et al., 2019).\n\nY-maze. This test measures spontaneous alternation as an index of working memory (Sarter et al., 1988). The maze is made of black-painted wood, and each arm is 25 cm long, 14 cm high, 5 cm wide, and positioned at equal angles. The mouse is placed at the end of one arm to move freely from side to side of the maze during an 8-min session. Videos recorded the sequence of entries during the whole time of the experiment and were analysed off-line. Entrance to each arm is scored when the mouse places the hind paws entirely in the zone. Alternation was defined as successive entries into the three arms on overlapping triplet sets. Consecutive triplets were analyzed, and alternate behaviour was calculated as the percentage of actual alternation (number of triplets with non-repeated entries) versus total alternation opportunities (total number of triplets), as described (Recinto et al., 2012; Yan et al., 2017).\n\nSpatial Y-maze. This test was used for spatial, novelty-seeking, and short-term memory assessment by measuring time spent in the novel arm (Hausrat et al., 2015; Biundo et al., 2018). As before, the maze was made of black-painted wood and each arm was 25 cm long, 14 cm high, 5 cm wide and positioned at equal angles. Each mouse was allowed to explore two arms of the Y-maze apparatus during the first trial (training) for 5 min. One hour later, the third arm was opened, and the mouse was returned to the same maze and allowed to explore all the three arms (testing). Visual cues were used to guide environment exploration, as described (Biundo et al., 2018). Animals with preserved cognition remember the previously blocked arm and they will enter it first on the second trial and spend more time exploring it. Distribution of mice and novel arms were balanced within each group. We cleaned the maze with 70% ethanol to remove olfactory cues between trials.\n\nRota-rod. Motor coordination was assessed with the rota-rod test, as described before (Fernandez et al., 1998). Briefly, mice were submitted to 1 min training session in the immobile apparatus. When the mouse falls, it is placed back into the rotating rod. Thereafter, mouse performance was tested in 5 min sessions every 15 min in 4 trials with increasing acceleration up to 40 rpm. The latency to fall off the rod in the final trial was measured and compared between groups.\n\nSocial behavior. Social behavior includes rewarding and motivational processes (Trezza et al., 2011; McCall and Singer, 2012). We studied social affiliation and social novelty/preference as described by others (Kaidanovich-Beilin et al., 2011). We placed each mouse in a cage with three compartments (one central and two lateral arms); in each compartment, we added a grid with one stranger mouse or an empty grid to assess social affiliation (intention to stay with the same species). We leave the mouse to explore for 10 minutes and record the time of direct interaction. Then, we cleaned the three chambers with ethanol (70%) to eliminate olfactory cues and placed the mice again in the center chamber. We include the previous stranger mice in the same arm (now named “familiar mouse”). In the empty space we include a new mouse (“stranger mouse”) and leave the animal free to explore and record the time of direct interaction.\n\nImmunocytochemistry was performed as described in detail before (Fernandez et al., 2012). A pre-treatment of 70% formic acid was used before incubation with anti-human Aβ antibody (1:50, Dako clone 6F/3D). Primary antibody was omitted as control. Confocal analysis was performed in a Leica (SP5 Direct, Germany) microscope. For plaque morphometry, 1-4 vibratome brain sections (50 μm, parietal cortex and hippocampus) were used to assess the density of Aβ plaques using Imaris software (Vs 9.0.2) (RRID:SCR_007370). Measurements were done as explained in detail elsewhere (Fernandez et al., 2012). Briefly, images were recorded using a 5X objective and were converted to gray scale to improve the contrast between signal and noise. All pictures were measured separately applying the same threshold. Areas were measured inside a reference circle in the hippocampus or parietal cortex with a standard size of 300 mm2. We then calculated the percentage of reference area occupied by Aβ plaques.\n\nTotal RNA isolation from brain tissue was carried out with Trizol (Life Technologies, USA) as described (Santi et al., 2017). For quantification, total RNA was isolated and transcribed using High Capacity cDNA Reverse Transcription Kit (ThermoFisher Scientific, Waltham, MA, USA, catalog number 4374966) following manufacturer’s instructions and 62.5 ng of cDNA was amplified using TaqMan probes for mouse IGF-1R and IR, and rRNA 18S as endogenous control. Cycling conditions were: UNG incubation: 50°C 2 minutes 1 cycle, Enzyme activation: 95°C 10 minutes 1 cycle, Denature: 95°C 15 seconds 40 cycles and Anneal/Extend: 60°C 1 minute, 40 cycles in a 7500 Real-time PCR system (Applied Biosystems). Relative mRNA expression was determined by the 2−ΔΔCT method (Pfaffl, 2001), and normalized to 18S rRNA levels. All probes were from Applied Biosystems: TaqMan Gene Expression Master Mix, catalog number 4369016 TaqMan Gene Expression assay, catalog number 4331182. Insulin receptor, Assay ID: Mm01211875_m1, FAM-MGB dye. Insulin like growth factor 1 receptor, Assay ID: 4331182 Mm00802831_m1, FAM-MGB dye. Eucaryotic 18S rRNA, Assay ID: Hs99999901_s1, VIC-MGB dye. At least five independent assays were carried out.\n\nThe number of animals for each experiment was calculated according to past experience with no hypothesis-driven outcomes, as these are observational studies. All animals in each group were included in analyses with no exclusion criteria applied a priori. Values were relativized compared to the control or baseline condition. Results are expressed as the average of the relative values obtained in each independent test (mean ± standard error) for each experiment and analyzed with GraphPad Prism 8.0 software (RRID:SCR_002798) (alternative open access program: R Program). Normality was confirmed using the Shapiro-Wilk normality test and equal variances with Levene’s test. Later, student’s t-test was used for comparison of two groups, or ANOVA for comparison of more than two groups with a Tukey or Sidak’s post-hoc analysis. Further details are explained in each figure. A statistically significant difference was considered when p<0.05.\n\n\nResults\n\nRecent publications in different models of downregulation of either insulin or IGF-I receptors in astrocytes have started to unveil specific actions of these receptors in this type of glial cells (Cai et al., 2018; Logan et al., 2018; Manaserh et al., 2019; Noriega-Prieto et al., 2021). We confirm that adult GFAP IR KO mice gradually show a depressive-like phenotype (Cai et al., 2018), as determined by the forced swim and the tail suspension tests. These alterations are seen in adult (>6 months old), but not younger mice (Figure 1A-B) (Zegarra-Valdivia et al., 2022). The existence of a depressive-like phenotype was reinforced by the observation that adult GFAP IR KO mice show disturbed responses to social novelty (Figure 1C), although not to social affiliation (Figure 1D). As determined in the open field test and elevated plus maze, GFAP IR KO mice did not show changes in anxiety levels either, which are frequently associated to depression (Figure 1E-F). These mice have intact cognition, as determined in the Barnes and Y maze tests assessing learning and memory (Figure 2A-C). In addition, GFAP IR KO mice did not show deficits in ambulation or motor coordination (Figure 2C-D).\n\nA, Adult (right histograms, control n= 6, GFAP IR KO n=7, t-test, t= 2.54; *p<0.05), but not young (left histograms, control n= 8, GFAP IR KO n=7, t-test, t= 1.26, p=0.22) GFAP IR KO mice show increased immobilization time in the tail suspension test (upper drawing), an indicator of a depressive-like behavior and reduced resilience to stress. B, Similarly, in the forced swim test (upper drawing), adult (right histograms, control n= 5, GFAP IR KO n=6, t-test, t= 2.5; *p<0.05, Welch`s correction), but not young (left histograms, control n= 5, GFAP IR KO n=6, t-test, t= 0.10; p=0.922) GFAP IR KO mice show increased depressive-like performance, with less time spent swimming. C, Social novelty, as measured by time spent with a novel partner vs a familiar one (upper drawing), was impaired in GFAP IR KO mice (control n= 10, GFAP IR KO n=12, t-test, t= 2.25; *p<0.05). D, Social affiliation, as determined by time spent with a stranger mouse vs an empty cage, was normal in GFAP IR KO mice (control n= 10, GFAP IR KO n=12, 2-way RM ANOVA, condition factor, F(1,20)=28.74; ***p<0.001, Sidak's multiple comparisons test, control familiar mice vs empty cage, ***p<0.001, GFAP IR KO novel vs familiar, **p<0.01). E, Time spent in the center of an open arena (upper drawing), a measure of novelty stress indicating levels of anxiety remained within control levels in adult GFAP IR KO mice (n=12 per group; t-test; t=0.77, p=0.445). F, Anxiety levels, as determined by time in the open arms of the elevated plus maze (upper drawing), are slightly, were normal in adult GFAP IR KO mice (control n= 21, GFAP IR KO n=24, t-test, t= 1.46, p=0.15). GFAP=glial fibrillary astrocytic protein.\n\nA, Adult GFAP IR KO mice performed similarly in the Barnes maze as compared to littermates, indicating intact spatial learning (n=9 per group, training days: 2-way ANOVA: F(1,36)=0.10, p=0.74; test day: t-test, Welch’s correction, t=0.65, p=0.53). B, Time spent in the novel arm of the spatial Y maze was similar to littermates in GFAP IR KO mice (n=9 per group, t-test, t=1.24, p=0.23). C, Number of spontaneous alternations in the arms of the Spontaneous Alternation Y maze, a measure was similarly unaltered in adult GFAP IR KO mice (n=9 per group, t-test, Welch’s correction, t= 0.67, p=0.51). D, No differences were observed in horizontal (left histograms) and vertical (right) activity in the open field arena was observed between experimental groups (n=12 per group, H: t-test, t=0.84, p=0.40; V: t-test, t= 0.05, p=0.95). E, Control littermates and GFAP IR KO mice show similar levels of motor coordination, as assessed in the rota-rod (control n=12, GFAP IR KO n=14, H: t-test, t=1.52, p=0.14). IR=insulin receptors, GFAP=glial fibrillary astrocytic protein.\n\nConversely, GFAP IGF-IR KO mice show specific impairments in spatial memory as assessed in the Barnes and Y mazes (Figure 3A-B), confirming previously observed deficits in cognition in these mice (Noriega-Prieto et al., 2021). However, working memory, as assessed by the alternation ratio in the Y maze, was intact (Figure 3C). These mice show normal social affiliation, whereas their preference for a novel partner was slightly impaired (Figure 3D-E). GFAP IGF-IR KO mice did not show mood disturbances either, as determined by time spent in the center of an open arena or in the open arms of the elevated plus maze (Figure 4A-B). GFAP IGF-IR KO show normal ambulatory behavior in the open field (Figure 4C), and in motor coordination tested in the rota-rod (Figure 4D).\n\nA, Spatial learning in the Barnes maze (upper drawing) was markedly affected in GFAP IGF-IR KO mice, showing significantly reduced memory (control n= 12, GFAP IR KO n=19, training days: 2-way ANOVA, time factor, F(3,122)=12.7; ***p<0.001, Sidak's multiple comparisons test, control vs GFAP 4th day of training, *p<0.05; test day: Mann-Whitney U: 21.5, ***p<0.001). B, Time spent in the novel arm of the Y maze (upper drawing), a measure of spatial memory, was reduced in GFAP IGF-IR KO mice (control n= 8, GFAP IGF-IR KO n=12, t-test, t= 2.26, *p<0.05). C, Number of spontaneous alternations in a Y maze, a measure of working memory (upper drawing), was similarly unaltered in adult GFAP IGF-IR KO mice (n=8 per group; t-test; t=0.98, p=0.342). D, Social affiliation, as determined by time spent with a stranger mouse vs an empty cage (upper drawing), was normal in GFAP IGF-IR KO mice (control n=9, GFAP IGF-IR KO n=8, 2-way RM ANOVA, condition factor, F(1,15)=19.13; ***p<0.001, Sidak's multiple comparisons test, control familiar mice vs empty cage, **p<0.01, GFAP IR KO familiar vs empty cage, *p<0.05). E, Social novelty, as measured by time spent with a novel partner (upper drawing), was impaired in GFAP IGF-IR KO mice (control n=9, GFAP IGF-IR KO n=8, 2-way RM ANOVA, condition factor, F(1,15)=11.18; **p<0.01, Sidak's multiple comparisons test, control novel mice vs familiar mice, *p<0.05, GFAP IGF-IR KO novel mice vs familiar mice, p=0.16). GFAP=glial fibrillary astrocytic protein.\n\nA, No differences in time spent in the center of an open field were observed between littermates and mutant GFAP IGF-IR KO mice (control n=28, GFAP IGF-IR KO n=22, t-test, t=0.73, p=0.46). B, No differences were observed in anxiety levels determined in the EPM between GFAP-IGF-IR KO and littermates (control n=16, GFAP IGF-IR KO n=15, t-test, t=0.48, p=0.63). C, No differences were observed in horizontal (left histograms) and vertical (right) activity in the open field arena was observed between GFAP IGF-IR KO mice and littermates (control n=28, GFAP IR KO n=22, H: t-test, t=0.61, p=0.54; V: t-test, t=0.12, p=0.90). D, Control and GFAP-IGF-IR KO mice show similar levels of motor coordination, as assessed in the rota-rod (control n=22, GFAP IR KO n=27, t-test, t=0.22, p=0.82).\n\nMice lacking IGF-IR in neurons show reduced AD-like pathology when cross-bred with a mouse AD model (Gontier et al., 2015), whereas mice lacking IR in neurons have not shown changes in AD-like pathology (Freude et al., 2009b). To analyze possible cell-dependent actions of these receptors in AD-like pathology, we crossed either GFAP IR KO or GFAP IGF-IR KO mice with APP/PS1 mice to obtain compound mutants and determined the impact of these receptors in memory loss associated to AD pathology seen in this mouse model. We observed that double GFAP IR KO/APP-PS1 mice presented significantly greater working memory loss compared to controls, as indicated by reduced spontaneous alternation in the Y maze (Figure 5A). In contrast, double GFAP IGF-IR KO/APP-PS1 showed enhanced cognition when compared to APP/PS1 mice, but no changes when compared to vehicle-treated GFAP IGF-IR/APP-PS1 mice (Figure 5B). Importantly, vehicle-treated control with preserved IGF-IR activity in astrocytes also showed enhanced cognition when compared to APP/PS1 mice (Figure 5B). Associated to greater memory loss we observed greater amyloid load in GFAP IR KO/APP-PS1 mice (Figure 5C), while in GFAP IGF-IR KO/APP-PS1 mice changes in amyloid plaque load were, again, control-dependent (Figure 5D). When compared to APP/PS1 mice, no changes were seen, but when compared to vehicle-treated controls, plaque load was increased. Of note, vehicle-treated controls show reduced plaque load when compared to APP/PS1 controls (Figure 5D).\n\nA, Performance in the working memory version of the Y maze was impaired in GFAP IR KO APP/PS1 mice (n=8 per group; One-way ANOVA, F=73.23; ***p<0.001; Tukey’s Multiple comparison test, APP/PS1 vs. GFP IR KO-APP/PS1: ***p<0.001, GFP IR Control-APP/PS1 vs. GFP IR KO-APP/PS1: ***p<0.001). B, Working memory determined in the Y maze remained unaltered in GFAP IGF-IR KO APP/PS1 mice and controls (n=8 per group; One-way ANOVA, F=2.9; p=0.07). C, Amyloid plaques in the parietal cortex and hippocampus in GFAP IR KO/APP-PS1 mice and controls. Representative photomicrographs showing amyloid plaques (red). Histograms show number of plaques/μm2 in three experimental groups (n=8 per group; One-way ANOVA, F=25.78; ***p<0.001; Tukey’s Multiple comparison test, APP/PS1 vs. GFP IR KO-APP/PS1: **p<0.01, GFP IR Control-APP/PS1 vs. GFP IR KO-APP/PS1: **p<0.001). D, Amyloid plaques in the parietal cortex and hippocampus in GFAP IGF-IR KO/APP-PS1 mice and controls does not show differences between groups. Representative photomicrographs showing amyloid plaques (red). Histograms show number of plaques/μm2 in the three experimental groups (n=8 per group; One-way ANOVA, F=1.35; p=0.32). IR=insulin receptors, GFAP=glial fibrillary astrocytic protein.\n\n\nDiscussion\n\nThe present results confirm and extend previous data of behavioral disturbances in GFAP IR KO or GFAP IGF-IR KO mice (Cai et al., 2018; Noriega-Prieto et al., 2021), and point to cell, receptor and context-specific actions of these receptors in the brain. These observations also indicate that insulin and IGF-I receptors in astrocytes play different roles in regulating memory and plaque formation in response to AD-like familial amyloidosis.\n\nAbsence of astrocytic IR led to deteriorated performance in mood-related tests without affecting cognitive tests such as the Y and Barnes mazes. The latter agrees with no changes in cognitive performance in the absence of IR in neurons (Plum et al., 2005), although more detailed studies are needed to determine the role of the neuronal IR in cognition, sociality and mood. Conversely, knock-down of IGF-IR in astrocytes affected performance in spatial memory tests and novelty-seeking such as the Barnes and Y mazes dependent on contextual clues, without affecting performance in the open field or elevated plus maze measuring mood traits. These mice showed normal working memory, though (Noriega-Prieto et al., 2021). Intriguingly, absence of IGF-IR in neurons alters mood and social interactions, together with cognitive disturbances (Zegarra-Valdivia et al., 2021; Fernandez de Sevilla et al., 2022). Finally, combined loss of IR and IGF-IR in all brain cells within specific regions results in both mood and cognitive disturbances (Soto et al., 2019). Thus, cell-specific actions of IR and IGF-IR receptors on mood and behavior appear the norm.\n\nReported discrepancies on the role of ILP receptors in the brain most probably arise from the varied experimental approaches used. This is true for both physiological and pathological processes. When the role of IGF-IR in brain proteostasis was determined, evidence was obtained using an heterozygous constitutive, whole body IGF-IR KO mouse bred in an APP/PS1 background (Cohen et al., 2009). This mouse showed reduced AD-related functional deficits but larger amyloid plaques. Additional confirmation of an involvement of IGF-IR in AD-like pathology was obtained using a homozygous neuronal-only tamoxifen-regulated IGF-IR KO mouse bred in an APP/PS1 background (Gontier et al., 2015). However, in this mouse, amyloid plaques and AD-related neuroinflammation were diminished, in agreement with previous observations in a Cre-dependent homozygous neuronal-only IGF-IR KO mouse bred in a mutant APP background (Freude et al., 2009b). This mouse also showed reduced amyloidosis and AD-related mortality, but no effects on other AD-related pathology were reported (Freude et al., 2009b). No noticeable effect of the absence of IR in these mice was observed either (Freude et al., 2009b).\n\nOur observations reinforce the notion that modification of AD-like pathology after manipulation of IR or IGF-IR activity in brain cells is highly dependent on experimental conditions. Thus, we observed increased plaque abundance and worsened working memory using the Y maze in double mutant GFAP IR KO/APP-PS1. This observation allows us to consider that astrocyte IR plays a protective role against AD-like pathology. However, when using GFAP IGF-IR KO/APP-PS1 mice, the situation is more complex. Working memory in the Y maze is improved in both double mutant GFAP IGF-IR/APP-PS1 mice, regardless of whether the IGF-IR was deleted, as vehicle control littermates show a similar enhanced performance in the Y maze. Conversely, while GFAP IGF-IR KO mice did not show changes in plaque load when compared to APP/PS1 controls, GFAP IGF-IR mice treated with vehicle show decreased plaque load. Therefore, we can conclude that the actions of IR and IGF-IR are highly dependent on the experimental model used and that in the case of IGF-IR, the control littermate group show changes when compared to control APP/PS1 mice, which poses a cautionary note on the interpretation of results.\n\nSeveral limitations should be stated. Although mouse models are successfully used to mimic human physiology and pathology, species-specific differences between mice and humans, should always be kept in mind when translating these observations. The reduced sample size in each experiment contributes to potential imprecision. Since bias in behavioral studies in experimental animals include sex of the experimenter performing the test, both male and female experimenters carried out these analyses. Together with the fact that mouse models of AD-like pathology, which are based in the genetic, least frequent type of AD, lack important aspects of the disease (most prominently, widespread neuronal loss), we consider that with the current available data, the role of ILP receptors in AD pathology remains undefined. Until better animal models of AD become available, and experimental approaches manipulating IR and IGF-IR activity are harmonized, we think this search should be re-formulated.\n\n\nData availability\n\nHarvard Dataverse: DATA SET - ASTROCYTE INSULIN AND INSULIN-LIKE GROWTH FACTOR I (IGF-I) RECEPTORS. https://doi.org/10.7910/DVN/Y7K97E (Zegarra-Valdivia et al., 2022).\n\nThis project contains the following underlying data:\n\n- APP-PS1 (para FIRKOTAPP) 10x ProjMax001.tif\n\n- APP-PS1(para BIRKOTAPP) 10x ProjMax001.tif\n\n- BIRKOTAPP Control 10x ProjMax001.tif\n\n- BIRKOTAPP KO 10x ProjMax001.tif\n\n- DATA SET - ASTROCYTE INSULIN AND INSULIN-LIKE GROWTH FACTOR I (IGF-I) RECEPTORS v.2.xlsx\n\n- FIRKOTAPP Control10x ProjMax001.tif\n\n- FIRKOTAPP KO 10x ProjMax001.tif\n\n- qPCR Data - Protocol.docx\n\n- qPCR InsR-IGF1R Ct values.xlsx\n\n\nReporting guidelines\n\nHarvard Dataverse: ARRIVE checklist for ‘Insulin and insulin-like growth factor-I receptors in astrocytes exert different effects on behavior and Alzheimer’s-like pathology’. https://doi.org/10.7910/DVN/Y7K97E.\n\nData are available under the terms of the Creative Commons Zero “No rights reserved” data waiver (CC0 1.0 Public domain dedication).",
"appendix": "Acknowledgements\n\nWe are thankful to M. Garcia for technical support.\n\n\nReferences\n\nBiundo F, Del Prete D, Zhang H, et al.: A role for tau in learning, memory and synaptic plasticity. Sci. Rep. 2018; 8: 3184. PubMed Abstract | Publisher Full Text\n\nCai W, Xue C, Sakaguchi M, et al.: Insulin regulates astrocyte gliotransmission and modulates behavior. J. Clin. Invest. 2018; 128: 2914–2926. PubMed Abstract | Publisher Full Text\n\nCohen E, Bieschke J, Perciavalle RM, et al.: Opposing Activities Protect Against Age Onset Proteotoxicity. Science. 2006; 313: 1604–1610. PubMed Abstract | Publisher Full Text\n\nCohen E, Paulsson JF, Blinder P, et al.: Reduced IGF-1 signaling delays age-associated proteotoxicity in mice. Cell. 2009; 139: 1157–1169. PubMed Abstract | Publisher Full Text\n\nDe Magalhaes Filho CD, Kappeler L, Dupont J, et al.: Deleting IGF-1 receptor from forebrain neurons confers neuroprotection during stroke and upregulates endocrine somatotropin 1. J. 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PubMed Abstract | Publisher Full Text\n\nShimizu T, Baba T, Ogawara M, et al.: Lifespan and glucose metabolism in insulin receptor mutant mice. J. Aging Res. 2011; 2011: 1–10. Publisher Full Text\n\nSoto M, Cai W, Konishi M, et al.: Insulin signaling in the hippocampus and amygdala regulates metabolism and neurobehavior. Proc. Natl. Acad. Sci. U. S. A. 2019; 116: 6379–6384. PubMed Abstract | Publisher Full Text\n\nTrezza V, Campolongo P, Vanderschuren LJ: Evaluating the rewarding nature of social interactions in laboratory animals. Dev. Cogn. Neurosci. 2011; 1: 444–458.\n\nYan T, He B, Wan S, et al.: Antidepressant-like effects and cognitive enhancement of Schisandra chinensis in chronic unpredictable mild stress mice and its related mechanism. Sci. Rep. 2017; 7: 6903. PubMed Abstract | Publisher Full Text\n\nZegarra-Valdivia JA, Santi A, Fernandez de Sevilla ME, et al.: Serum Insulin-Like Growth Factor I Deficiency Associates to Alzheimer’s Disease Co-Morbidities. J. Alzheimers Dis. 2019; 69: 979–987. PubMed Abstract | Publisher Full Text\n\nZegarra-Valdivia JA, Fernandes J, Esparza J, et al.: Interoceptive Information of Physical Vigor: Orexin Neurons Gauge Circulating IGF-I for Motivational Motor Output. bioRxiv:2021.2005.2025.445442. 2021.\n\nZegarra-Valdivia JA, Fernandez A, Martinez-Rachadell L, et al.: Data Set - Astrocyte Insulin and Insulin-Like Growth Factor I (IGF-I) Receptors. [Data] Harvard Dataverse, V5.2022. Publisher Full Text"
}
|
[
{
"id": "141517",
"date": "25 Jul 2022",
"name": "Andre Kleinridders",
"expertise": [
"Reviewer Expertise insulin signaling",
"brain metabolism",
"behavior",
"diabetes",
"obesity"
],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nZegarra-Valdivia et al., Insulin and insulin-like growth factor-I receptors in astrocytes exert different effects on behavior and Alzheimer´s-like pathology\nIn their manuscript, Zegarra-Valdivia and colleagues investigate the effect of Insulin receptor (IR) and IGF-1 receptor (IGF-1R) deficiency in astrocytes on behavior using a tamoxifen-inducible knockout approach. The authors show that time-dependent IR deletion in GFAP astrocytes (GFAP IR KO mice) develop mood alterations, while cognition is not impacted. Yet, deficiency of IGF-1R in astrocytes causes a more severe behavioral phenotype with decreased cognition and depressive-like behavior. When these mouse models were bred with APP/PS1 mice, a model of familial Alzheimer´s disease (AD), the lack of IR worsened the phenotype, while deficiency of IGF-1R attenuated this phenotype. Yet, lox/lox animals showed the same phenomenon, as KO mice, questioning the effect of IGF-1R in this scenario. It remains elusive, why the observed phenotype is present, as no molecular insights were given.\nOverall the finding in this manuscript is very interesting and expands knowledge about the effect of insulin/IGF signaling in the brain on behavior. It addresses an important question, whether deletion of IR or IGF-1R in astrocytes impact behavior when deleted in adulthood. As most humans acquire insulin resistance while aging and not in their youth, these mouse models exhibit an important feature of age-associated brain insulin resistance and shed light into late-term complications. Thus the use of a tamoxifen-inducible knockout (KO) model is appropriate to gain more insights into time-dependent effects of IR and IGF-1R deficiency in astrocytes.\nFollowing issues with their presented data and conclusions should be addressed, to help the unfamiliar reader to better understand their results and compare it to already published literature.\nThe authors need to show tamoxifen-induced deletion deficiency, to better understand their acquired data. The behavioral traits, which have been investigated, are impacted by various brain regions. Is the IR or IGF-1R efficiently deleted in astrocytes in those regions? Is IR upregulated in IGF-1R KO mice and vice versa?\n\nDoes the lack of IR or IGF-1R alters blood glucose levels, as hyper- and hypoglycemia exhibit profound effects on brain physiology?\n\nDo these mice exhibit neuroinflammation, oxidative stress or even apoptosis? Markers such as GFAP, Iba, lipid peroxidation etc. should be investigated to gain insights into potential mechanisms.\n\nIt has been shown that lack of IR or IGF-1R in astrocytes alters brain energy metabolism. Do mice with tamoxifen-induced IR and IGF-1R deletion in astrocytes show a similar effect?\n\nWhy does IR deficiency worsens the phenotype in APP/PS1 mice, yet IGF1R deletion not? What are proposed mechanisms?\n\nWhy do APP/PS1 lox/lox IGF-1R mice show a different phenotype compared to APP/PS1 mice? Why do these control mice exhibit the same phenotype as APP/PS1 IGF-1R KO animals? Here more research is needed.\n\nCould the authors please state how many males and females participated for each experiment? Does ‘sex-balanced manner’ stand for an equal number of males and females and were there differences between the different tests? Is there a sex-specific difference, as it has been shown for conditional KO animals?\nMinor comments:\nIn Figure 1A and B the authors show results of the Mousetail suspension test and Forced swimming test which were not described. The authors should add the test description in the method section.\n\nFigure 5A and C do not show single data points compared to other figures. Please change 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? 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? Yes",
"responses": [
{
"c_id": "8995",
"date": "30 Nov 2022",
"name": "Ignacio Torres Aleman",
"role": "Author Response",
"response": "\"The authors need to show tamoxifen-induced deletion deficiency, to better understand their acquired data. The behavioral traits, which have been investigated, are impacted by various brain regions. Is the IR or IGF-1R efficiently deleted in astrocytes in those regions? Is IR upregulated in IGF-1R KO mice and vice versa?\" Answer: Both mouse models have been described in detail by us in previous publications: Garcia-Caceres et al Cell 2016, Hernandez-Garzon et al Glia 2016, and Noriega-Prieto J Neurosci 2021. Efficiency of Cre recombination after tamoxifen injection was shown in those publications. We did not examine all brain regions for deletion of IR or IGF-I respectively, only cortex and hippocampus. These two regions are involved in all tested behaviors. As indicated in the Glia 2016 publication, brain IR levels in IGF-IR KO mice were similar to wild types and these mice responded normally to insulin. Conversely, brain IGF-IR levels in IR KOs were similar to wild type levels, and responded normal to IGF-I (Fernandez et al, PNAS 2022). We now address these points in the corrected manuscript, adding these supporting references. \"Does the lack of IR or IGF-1R alters blood glucose levels, as hyper- and hypoglycemia exhibit profound effects on brain physiology?\" Answer: Indeed, peripheral glucose levels will impact on brain physiology. The pattern of changes in blood glucose in these mice is time and sex-dependent. Young, but not middle-aged GFAP IR KO mice show hyperglycemia (Fernandez et al, PNAS 2022) and glucose intolerance (Garcia-Caceres et al, Cell 2016). Female, but not male GFAP-IGF-IR KO mice show glucose resistance (unpublished). We now introduce these data to nuance the discussion. \"Do these mice exhibit neuroinflammation, oxidative stress or even apoptosis? Markers such as GFAP, Iba, lipid peroxidation etc. should be investigated to gain insights into potential mechanisms.\" Answer: Indeed, all these factors could help explain the observed phenotypes. We only have analyzed in greater detail GFAP-IR KO mice, that show oxidative stress (Fernandez et al PNAS 2022). We have included new comments in the discussion to address this point. \"It has been shown that lack of IR or IGF-1R in astrocytes alters brain energy metabolism. Do mice with tamoxifen-induced IR and IGF-1R deletion in astrocytes show a similar effect?\" Answer: Yes, both IR (Fernandez et al, PNAS 2022) and IGF-IR (Hernandez-Garzon et al, Glia 2016, and in preparation) show changes in brain glucose metabolism. This comment has also been introduced in the discussion. \"Why does IR deficiency worsens the phenotype in APP/PS1 mice, yet IGF1R deletion not? What are proposed mechanisms?\" Answer: This is a key question. The observational data that we now present does not intend to answer it and will involve a variety of mechanistic studies. We wanted to publish these observations to illustrate that the effects of IR and IGF-IR in the APP/PS1 mouse depend on many variables not accounted for in previous studies. This was our solely aim. Analysis of underlying mechanisms in these specific models will in all probability unveil mechanisms related to these specific models. Our point is that with the current available approaches is premature to establish a role of IR and IGF-IR in Alzheimer disease. \"Why do APP/PS1 lox/lox IGF-1R mice show a different phenotype compared to APP/PS1 mice? Why do these control mice exhibit the same phenotype as APP/PS1 IGF-1R KO animals? Here more research is needed.\" Answer: Indeed, more research is needed if we want to answer these key points. We do not have any evidenced-based explanation. Floxing the IGF-IR gene may have unknown functional consequences. We believe that this type of observations are not enough to support a deleterious role of IGF-IR in Alzheimer disease, which has been until now widely held. \"Could the authors please state how many males and females participated for each experiment? Does ‘sex-balanced manner’ stand for an equal number of males and females and were there differences between the different tests? Is there a sex-specific difference, as it has been shown for conditional KO animals?\" Answer: Both sexes were used in similar proportions but not 50/50 in all experiments, in some 40/60 ratio was used. There may be sex differences (as seen in various traits in these KOs), but we did not consider necessary to use larger number of animals (also for ethical considerations) since our results already document that these models are not appropriate to reach robust conclusions about the role of IR/IGF-IR in mouse model of AD. Minor comments: \"In Figure 1A and B the authors show results of the Mouse tail suspension test and Forced swimming test which were not described. The authors should add the test description in the method section.\" Corrected as indicated (see new methods). Apologies for the oversight. \"Figure 5A and C do not show single data points compared to other figures. Please change accordingly.\" Corrected as indicated. Again, apologies for the oversight."
}
]
},
{
"id": "153403",
"date": "04 Nov 2022",
"name": "Susana Cardoso",
"expertise": [
"Reviewer Expertise Brain energy metabolism",
"Mitochondria",
"brain signaling pathways"
],
"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 by Zegarra-Valdivia and co-workers intended to elucidate the role of insulin receptors (IR) and insulin-growth factor 1 receptor (IGF-1R) in astrocytes. In particular, the authors had a specific interest in investigating the role of both receptors in mood and cognitive traits of young and older mice as well as in an AD-like context. To accomplish their goals, the authors used different KO models and performed the appropriate behavioral analysis. Overall, the rational of the study looks interesting, but the data presented seems to be quite preliminary to present conclusive findings.\nPlease consider addressing the following points during the revision process:\nStarting with the results, it is quite visible that the number of animals used to perform the forced swim test is not the same as the ones submitted to the other tests (Fig. 1). Do the authors have any justification for this? Such discrepancy can introduce significant bias on the data analysis.\n\nAlso, why is the number of animals used to evaluate cognitive behavior in IGF-1R KO (Fig.3) so different from the number of animals used to evaluate mood homeostasis (Fig.4)?\n\nThe graphs of Figure 5A and 5C do not have the individuals’ data points. Please check this.\n\nAlthough the authors performed a reasonable detailed description of the methods, there are some points missing. It is mentioned that the authors used young and older mice of the IR KO genotype, but is not clear what was the age of the mice used from the IGF-1R genotype? And why were two age points for the IR-KO mice used and only one age point for the IGF-1R KO mice?\n\nAlso, evidence should be provided that the experimental mice are really KO in astrocytes as well as that the KO is not all-body.\n\nIn methods qPCR technique is described, but the manuscript does not have any mRNA data. Please check this.\n\nFigure 5 should have data from control animals per se. The results presented in Figure 5B and 5D are quite difficult to understand. What are the genotypic differences between the APP/PS1 mice and the GFP-IGF-1R Control-APP/PS1 mice?\n\nIs the work 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?\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": "8996",
"date": "30 Nov 2022",
"name": "Ignacio Torres Aleman",
"role": "Author Response",
"response": "\"Starting with the results, it is quite visible that the number of animals used to perform the forced swim test is not the same as the ones submitted to the other tests (Fig. 1). Do the authors have any justification for this? Such discrepancy can introduce significant bias on the data analysis.\" Answer: Thank you for your comment. Depressive-like behavior in GFAP-IR KO mice was already reported by Cai et al (JCI 2018). These authors described the presence of mood alterations in young female GFAP-IR KO mice while males appear to have only minor deficits. The purpose of our study is to compare this model irrespective of sex differences in the context of AD as the role of IR in this disease is controversial. For this reason, we first confirmed that GFAP-IR KO mice (when both sexes are pooled together) show mood disturbances. In our hands, we found these disturbances in adult mice only (>6 months old) while Cai et al found them already in 4 month old mice, probably because we pooled together both sexes. Since we just wanted to confirm observations by others, we did not use more animals. In the rest of tests, no previous information was available, and for this reason we used more animals. In the case of the EPM test we used many more animals because Cai et al reported increased anxiety (without using the EPM test) in GFAP IR KO mice, but we did not see changes in this behavioral trait. \"Also, why is the number of animals used to evaluate cognitive behavior in IGF-1R KO (Fig.3) so different from the number of animals used to evaluate mood homeostasis (Fig.4)?\" Answer: Figure 3: in cognitive tests we obtained significant differences using samples sizes of 8-12 animals/group. Since in GFAP-IR KO mice we had detected mood disturbances, we wanted to confirm that in GFAP IGF-IR KO mice mood is not altered (Figure 4). For this reason, we increased sample size to be sure that it was not a problem of sample size. It is quite intriguing that mice lacking IR in astrocytes show mood disturbances but intact cognition while mice lacking IGF-IR in astrocytes show altered cognition and intact mood. \"The graphs of Figure 5A and 5C do not have the individuals’ data points. Please check this.\" Answer: Corrected. Apologies for the oversight. \"Although the authors performed a reasonable detailed description of the methods, there are some points missing. It is mentioned that the authors used young and older mice of the IR KO genotype, but is not clear what was the age of the mice used from the IGF-1R genotype? And why were two age points for the IR-KO mice used and only one age point for the IGF-1R KO mice?\" Answer: Young (< 6 months old) and adult (>6 months old) mice were used in both genotypes. This is now stated in the manuscript (Results, first subheading, second paragraph). The other part of this question has already been answered above. \"Also, evidence should be provided that the experimental mice are really KO in astrocytes as well as that the KO is not all-body.\" Answer: We copy the answer already given to the other reviewer addressing this point: Both mouse models have been characterized by us in different publications: Garcia-Caceres et al Cell 2016, Hernandez-Garzon et al Glia 2016, and Noriega-Prieto J Neurosci 2021. Efficiency of Cre recombination after tamoxifen injection was shown in those publications. We did not examine all brain regions for deletion of IR or IGF-I respectively, only cortex and hippocampus. These two regions are involved in all tested behaviors. As indicated in the Glia 2016 publication, brain IR levels in IGF-IR KO mice were similar to wild types and these mice responded normally to insulin. Also brain IGF-IR levels in IR KOs were similar to wild type levels, and responded normal to IGF-I (Fernandez et al, PNAS 2022). We now address these points in the corrected manuscript, adding these supporting references. \"In methods qPCR technique is described, but the manuscript does not have any mRNA data. Please check this.\" Answer: Thank you for the comment. In a draft version of the manuscript we were including mRNA levels in both genotypes, that is the reason for this error. We have deleted this subheading. \"Figure 5 should have data from control animals per se. The results presented in Figure 5B and 5D are quite difficult to understand. What are the genotypic differences between the APP/PS1 mice and the GFP-IGF-1R Control-APP/PS1 mice?\" Answer: Two types of controls are included in this Figure; APP/PS1 and APP/PS1x GFAP IGF-IR littermates. APP/PS1 mice have extensively been shown (including our previous publications) to present deficits in the Y maze and Abeta plaques. We agree that the results are quite difficult to explain, but they illustrate our main point: these mouse models are not adequate to ascribe a role for IGF-IR (or IR) in AD since the results are highly dependent on the model used. To our knowledge, previous results compared littermates and KOs in an APP/PS1 background, without including APP/PS1."
}
]
}
] | 1
|
https://f1000research.com/articles/11-663
|
https://f1000research.com/articles/10-604/v1
|
19 Jul 21
|
{
"type": "Data Note",
"title": "Data Note: COVID-19, social distancing, and pipeline vandalism in Nigeria",
"authors": [
"P. N. Onwuachi-Iheagwara",
"B.I Iheagwara",
"B.I Iheagwara"
],
"abstract": "We present a dataset of the monthly cases of pipeline vandalism in Nigeria from January 2015 to January 2021. Data used in this study were collated from the Monthly Financial and Operations Reports (MFOR) of the Nigeria National Petroleum Corporation (NNPC). Each MFOR provides cases of pipeline vandalism during a 12-month span from five key locations; Mosimi, Kaduna, Port Harcourt, Warri, and Gombe. Recorded incidences of pipeline vandalism from these locations were summed and assembled into five groups; namely: historical data, prior-COVID-19, COVID-19 lockdown, and post-COVID-19 lockdown. The data were grouped based on dates. These dates were January 2015 to July 2019, August 2019 to January 2020, February 2020 to July 2020, and August 2020 to January 2021 respectively. The historical data were further sub-divided into four sub-groups based on the deployment (May 2016) of sophisticated weapons, satellite imagery, and geographical information system into the security apparatus to checkmate pipeline vandalism. The four sub-groups are sub-group A (one-year before deployment), sub-group B (the year of deployment), sub-group C (one-year after deployment), and sub-group D (two-years after deployment). The dates span for each sub-group is May 2015-April 2016, May 2016-April 2017, May 2017-April 2018, and May 2018-April 2019 respectively. After the deployment of GIS devices in May 2016, the accumulated national number of pipeline vandalism cases declined from 400 cases in January 2016 to 293 in February 2016, and 259 cases in March 2016 as opposed to 60, 49, and 94 cases in the same months in 2017; but over the years, 2017 to 2021 these methods have proved less effective, and cases of pipeline vandalism have risen once more. Similar changes in the number of cases and patterns were observed during the COVID-19 movement restrictions. From the dataset, it can be seen that COVID-19 influenced incidences of pipeline vandalism.",
"keywords": [
"COVID-19",
"pipeline vandalism",
"restriction on movement",
"NNPC pipelines",
"pipes"
],
"content": "Introduction\n\nProduct theft and vandalism of national pipelines are recurring challenges faced by the Nigeria National Petroleum Corporation (NNPC).1,2 During the COVID-19 pandemic, movements were restricted. It is desirable to determine if a significant difference exists in the incidence of pipeline vandalism of Nigerian oil pipelines during the COVID-19 pandemic. This dataset provides that information.\n\n\nMethod\n\nIncidences of vandalism of pipeline are ascribed in the Monthly Financial and Operations Reports (MFOR) of the Nigeria National Petroleum Corporation (NNPC).5 In this research, data from the NNPC MFOR from January 2015 to January 2021 were used. These monthly reports are available for free download by the public from the NNPC website link (NNPC; https://www.nnpcgroup.com).3 The information abstracted from the NNPC MFOR were number of cases of pipeline vandalism per month, month of vandalism, and year of vandalism.\n\nThe data were grouped into four groups, namely:\n\n1. Historical data –1st January 2015 to 31st July 2019.\n\n2. Prior COVID-19 data – 1st August 2019 to 31st January 2020.\n\n3. COVID-19 data – 1st February 2020 to 31st July 2020.\n\n4. Post COVID-19 data – 1st August 2020 to 31st January 2021.\n\nFurthermore, we obtained information and dates of major events that may be considered possible external stimuli in this analysis. This information was collected from national and regional newspapers and web-based publications, and web pages.\n\nThese are:\n\n• May 2016, incorporation and deployment of sophisticated weapons, use of satellite images and geographical information system (GIS) into the security apparatus to ensure vandalism is contained, the setting up of a pipeline security force to stamp out the menace, and the formation of the Trans-National Organized Crime (TNOC) with regional allies to fight against the proliferation of Small Arms and Light Weapons.4 This was a welcome development as the area under physical patrol were massive.\n\n• The onset of COVID-19 in December 2019 and the declaration of COVID-19, on 30th January 2020, as a Public Health Emergency of International Concern by WHO (World Health Organization), and the upgrade to a pandemic by the 11th of March 2020.\n\n• In Nigeria, the pre-lockdown commenced from 28th February – 29th March, 2020, and was 31 days in duration. The lockdown, in total 35 days; was from 30th March to 3rd May, 2020, and’easing up’ of 73 days, 5th May – 15th July, 2020.\n\nThis information was mainly used in the interpretation of the plot of monthly cases of pipeline vandalism vs. time in month/year (Figure 1).\n\nThe May 2016 event (from a cursory glance of Figure 1) had a great impact on cases of pipeline vandalism. Thus, the historical data were further divided into four sub-groups to capture the possible influence of the deployment in May 2016 by the Nigerian government on pipeline vandalism.4\n\nThe four sub-groups of the historical data are:\n\n1. Sub-group A (one-year before deployment; 1st May 2015-30th April 2016),\n\n2. Sub-group B (year of deployment; 1st May 2016-30th April 2017),\n\n3. Sub-group C (one-year after deployment; 1st May 2017-30th April 2018),\n\n4. And sub-group D (two-years after deployment; 1st May 2018-30th April 2019).\n\nPrior to the deployment, the pipeline security method involved the active patrol in pipeline installation by security agents using patrol vehicles. Another method adopted by past administration was the involvement of local militia leaders in delicate but dangerous and remote locations. Subsequent to the deployment a combination of the active patrol of pipeline installation by security agents and GIS are used; in addition to a reversal of the policy on the use of local militia.4\n\n\nSoftware used in the data analysis\n\nThe MS office Excel 2013 with the Analysis ToolPak add-in were used. In all statistical analysis in this project, an alpha = 0.05 as the significance threshold was set, and a null and an alternative hypothesis were established. This means that the null hypothesis would be rejected if the p-value is less than or equal to 0.05 and the alternative hypothesis would be accepted.\n\n\n\n• Null hypothesis: There is no significant difference between the mean case of pipeline vandalism incidences prior, during, and post COVID lockdown.\n\n• Alternative hypothesis: There is a significant difference between the mean case of pipeline vandalism incidences prior, during, and post COVID-19.\n\nFor the group data, the total incidences during the time frame covered by the group, average, and standard deviation were established. The grouped data were subjected to an ANOVA analysis, and a time series analysis was undertaken after the data were smoothed using a moving average.\n\nFor the sub-groups, the total cases in each subgroup, the mean, and the standard deviation were calculated. A null and an alternative hypothesis were set.\n\n• Null hypothesis: There is no significant difference between the mean case of pipeline vandalism incidences prior, during, and post the deployment.\n\n• Alternative hypothesis: There is a significant difference between the mean case of pipeline vandalism incidences prior, during, and post the deployment.\n\nThe sub-grouped data were also subjected to an ANOVA analysis, and a time series analysis (after the data were smoothened by moving average).\n\nThird, it was observed that fewer cases of vandalism occurred in the historical sub-group B than in any other subgroup with major improvements six months after the deployment in May 2016. The six-month lag may be the “learning/training and implementation phase” after the media announcements and deployment. In the COVID-19 pandemics groups, the lockdown period presented fewer cases of pipeline vandalism. It was noted that periods of renovations of the methodologies used to checkmate the activities of vandals and uncompromising movement restrictions favoured a reduction in cases of pipeline vandalism. From a security viewpoint, it was therefore desirous to determine if greater success would be attributed to the use of either of the two methods (Figure 2). This would enable the government to design a more winning approach to vandalism.\n\n\nDataset validation\n\nSeasonal confounds\n\nThere are two principal seasons, the wet rainy season and the hot dry season in Nigeria. Pipeline vandalism takes place in remote locations on isolated, rural roads and footpaths; not readily accessible during adverse weather conditions. We, therefore, assume that the rainfall affects the number of cases of pipeline vandalism. However, rainfall patterns are fairly predictable and torrential rainfall occurs in the mid of the rainy seasons. Seasonal confounds were eliminated by comparing data for the same months in each group. This implies that the rainfall season data (in one group) were compared only with the rainfall season data (in another group); with similar arguments for the dry season data.\n\nData points in each group or sub-group\n\nFor all analysis, the number of data points was of uniform length to reduce any possible bias due to unparalleled data points.\n\nIn each COVID-19 group (prior, during, and post COVID-19 lockdown groups) the number of data points were six. In the four sub-groups of the historical data (i.e., sub-group A, sub-group B, sub-group C, and sub-group D) each sub-group had 12 data points.\n\nThe data was assembled over an even interval and ordered chronologically with equal time frequency.\n\nExclusion of data\n\nAll cases/incidences of vandalism of pipelines that fall before or after the time frame under review (1st January, 2015 to 31st January, 2021) as ascribed in the MFOR were removed from the analysis.\n\nOther assumptions made\n\nThe destruction of these pipelines has been a scourge on the national petroleum industry in Nigeria since time immemorial,1 two groups of people disrupt pipelines in Nigeria namely; activists, radicals, and militants, to make political statements, and thieves with the sole purpose to gain illegal possession of the fluids.\n\nThe former, makes political statements before any attempted disruptions, often to inform the government and allow negotiation for the fulfillment of their demands; the latter do not. During the lockdown, no activists, radicals, and militants made any political statement; so, we can assume they also heeded the order to isolate and social distance. We therefore attributed all pipeline vandalism during the COVID-19 lockdown period to thieves.\n\n\nResults\n\nOur dataset shows the monthly cases of pipeline vandalism from 2015 to 2021.5 It shows the variations in cases before and after the use of GIS for monitoring. It also revealed the changes in pre-COVID-19, post-COVID-19, and during the pandemic.\n\nThis dataset ensures the easy availability of this information for the general public.\n\n\nData availability\n\nHarvard Dataverse. Effects of COVID-19 on pipeline vandalism in Nigeria, West Africa. DOI: https://doi.org/10.7910/DVN/8X5KKB.5\n\nThis project contains the following underlying data:\n\nDataset Data for: Effects of COVID-19 on pipeline vandalism ingested files:\n\n• Original data.tab. (Contains the unfiltered data from the NNPC reports, with cases of pipeline vandalism tabulated by month and year.).\n\n• ANOVA-Historical subgroups.tab. (Two sheets. One; (MasterDataSheet) contains the original data divided into the four groups and a second (Historical sub-groups) preliminary analyses on the sub-groups).\n\n• ANOVA-COVID-19 groups.tab. (ANOVA analysis of COVID-19 group (prior, during, and post COVID-19 lockdown groups)).\n\n• Graph-subgrpB-and-6-months lockdown.tab. (Comparative analysis of key periods – 1st February-30st July 2017 and 1st February-30st July 2020).\n\n• Time series analysis -COVID-19 groups.tab. (Time series analysis of COVID-19 group (prior, during, and post COVID-19 lockdown groups) smoothening with moving average).\n\n• Time series analysis-Historical subgroups.tab. (Time series analysis of historical subgroups with smoothening by moving average).\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\nMmeje DU, Bello A, Mohammed UD: Investigation of Pipeline Vandalism and Its Implications on Business Activities in Nigeria. Journal of Resources Development and Management www.iiste.org. 2017; 2422-8397. An International Peer-reviewed Journal, Vol. 38, 2017.\n\nOkoli A, Chukwuma, Orinya S: Oil Pipeline Vandalism and Nigeria’s National Security. Global Journal of human social science Political Science. 2013; Volume 13 Issue 5 Version 1.0 Year 2013, Online ISSN: 2249-460x & Print ISSN: 0975-587X\n\nThe NNPC group. Reference Source\n\nBuhari’s Milestones in One Year – Presidency.Reference SourceDownloaded 10th May 2021\n\nOnwuachi-Iheagwara N: Data for: Effects of COVID-19 on pipeline vandalism in Nigeria, West Africa. Harvard Dataverse, V1, UNF:6:oZ02Sff1pmG9M3ThLAQNFw== [fileUNF]. 2021. Publisher Full Text"
}
|
[
{
"id": "89908",
"date": "28 Jul 2021",
"name": "Alessandro Rovetta",
"expertise": [
"Reviewer Expertise Infodemiology",
"Infoveillance",
"Public Health",
"Statistics"
],
"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 comments:\nThis paper investigates the impact of anti-COVID-19 restrictive measures on the vandalism pipeline by analyzing the time series of the incidence of the phenomenon from January 2015 to January 2021. To do this, an ANOVA is adopted after the data has been processed via a moving average. The authors conclude that COVID-19 influenced the incidences of pipeline vandalism. However, at present, there are numerous critical issues to be addressed before proceeding with indexing.\n\nMajor comments:\n1) Section: Introduction. This section needs some additions to represent a complete background and contextualize the paper in the current scenario. Specifically, I suggest briefly discussing:\n1.1. The incidence of pipeline vandalism in Nigeria and the related problems and damage (providing appropriate references)\n1.2. The usefulness of this study (i.e., what kind of information can this type of analysis return regarding pipeline vandalism? Why is it relevant to evaluate the impact of anti-pandemic restrictions on the phenomenon?)\n1.3. The significance of the paper for future research (i.e., how can this data note help government authorities deal with pipeline vandalism?)\n2) Section: Methods.\n\n2.1. “These monthly reports are available for free download by the public from the NNPC website link (NNPC; https://www.nnpcgroup.com)” To facilitate reproducibility, I kindly ask the authors to provide a more precise URL or path description to derive this dataset. Thank you.\n2.2. “The data were grouped into four groups, namely: [...]” I strongly suggest motivating those subdivisions in detail. In particular, based on what criteria and purpose were these groups formed?\n2.3. “May 2016, incorporation and deployment of sophisticated weapons, use of satellite images and geographical information system (GIS) into the security apparatus to ensure vandalism is contained, the setting up of a pipeline security force to stamp out the menace, and the formation of the Trans-National Organized Crime (TNOC) with regional allies to fight against the proliferation of Small Arms and Light Weapons. 4” . Reference 4 refers to a web page called “Oil and Gas 360.” Therefore, I kindly ask if it is possible to provide a more specific URL or refer to an academic source.\n2.4. “In Nigeria, the pre-lockdown commenced from 28th February – 29th March, 2020, and was 31 days in duration. The lockdown, in total 35 days; was from 30th March to 3rd May, 2020, and easing up’ of 73 days, 5th May – 15th July, 2020.” This information needs a reference.\n2.5. “The May 2016 event (from a cursory glance of Figure 1) had a great impact on cases of pipeline vandalism.” Figure 1 shows that the decreasing trend has occurred since July 2016. Therefore, it is necessary to argue the causal nature of the association made and discuss the presence of a time-series lag.\n2.6. “In all statistical analysis in this project, an alpha = 0.05 as the significance threshold was set, and a null and an alternative hypothesis were established. This means that the null hypothesis would be rejected if the p-value is less than or equal to 0.05 and the alternative hypothesis would be accepted.” Using a simple threshold for significance analysis is misleading (Amrhein et al. (20171), Greenland et al. (20162)). P-values should be used - at best - as a graded measure of the strength of evidence against the null hypothesis. Therefore it is necessary to report the P-values in full (if this is not possible, it is advisable to provide an additional file). Furthermore, P-values are unsuitable for measuring the intensity of a phenomenon (e.g., very weak but statistically significant phenomena can occur (Schober et al. (20183))). Therefore I suggest introducing quantifiers into the analysis (e.g., percentage differences).\n2.7. “The sub-grouped data were also subjected to an ANOVA, and a time series analysis (after the data were smoothened by moving average).” ANOVA analysis requires the verification of a certain number of assumptions, as explained here. Therefore, how the latter has been verified must be described in detail. Moreover, it is necessary to specify the type of ANOVA adopted. Finally, the amplitude of the moving average must be specified.\n2.8. A time-series analysis requires the verification of the absence/presence of previous trends. Therefore, I suggest adding this essential control.\n2.9. “Pipeline vandalism takes place in remote locations on isolated, rural roads and footpaths; not readily accessible during adverse weather conditions.” Since this sentence justifies a fundamental assumption, I suggest motivating it with a source.\n\nMinor comments:\nm1) Section: Method. “The onset of COVID-19 in December 2019 and the declaration of COVID-19, on 30th January 2020, as a Public Health Emergency of International Concern by WHO (World Health Organization), and the upgrade to a pandemic by the 11th of March 2020.” This sentence needs a reference. I can suggest this one here.\n\nm2) Section: Method. “During the lockdown, no activists, radicals, and militants made any political statement; [...]” It would be appropriate to justify this sentence with a source.\n\nIs the rationale for creating the dataset(s) clearly described? Partly\n\nAre the protocols appropriate and is the work technically sound? Partly\n\nAre sufficient details of methods and materials provided to allow replication by others? No\n\nAre the datasets clearly presented in a useable and accessible format? Partly",
"responses": [
{
"c_id": "7071",
"date": "11 Jan 2023",
"name": "N Onwuachi-Iheagwara",
"role": "Author Response",
"response": "1) Section: Introduction. This section needs some additions to represent a complete background and contextualize the paper in the current scenario. Specifically, I suggest briefly discussing: 1.1. The incidence of pipeline vandalism in Nigeria and the related problems and damage (providing appropriate references) Response: Oil spillage is associated with oil pipeline destruction. The destruction of pipelines leads to several environmental problems; this includes fresh and seawater pollution, air pollution, chemical pollution, soil and land pollution [1]. This also makes most agricultural practice unsustainable with an associated decline in fish farming in populated waters, biodiversity depletion [3], loss of habitat and loss of ecological and security systems [2,3,4]. References: Mmeje, David Uchechukwu, Bello Ayuba, U.D. Mohammed. Investigation of Pipeline Vandalism and Its Implications on Business Activities in Nigeria. Journal of Resources Development and Management, 2017 Vol.38, pp.69-81. ISSN 2422-8397 www.iiste.org Sanusi Aishatu ,Onovo Josiah Chukwudi and Isa Hauwa’u . The Environmental Impact of Pipeline Vandalism – A Challenge to Biodiversity in Port Harcourt, Area of Rivers State, Nigeria. Int’l Journal of Advances in Chemical Engg., & Biological Sciences (IJACEBS) Vol. 3, Issue 1 (2016) ISSN 2349-1507 EISSN 2349-1515 Nwagboso, C.I. Security Challenges and Economy of the Nigerian State (2007 – 2011), American International Journal of Contemporary Research, 2012 Vol. 2 No. 6, 244-.258. E. Ugwuanyi, “Steaming Vandalism Theft in Downstream Sector” in A.C. Okoli and S. Orinya, “Oil Pipeline Vandalism and Nigeria’s National Security” (2013) Global Journal of Human Social Science 1.2. The usefulness of this study (i.e., what kind of information can this type of analysis return regarding pipeline vandalism? Why is it relevant to evaluate the impact of anti-pandemic restrictions on the phenomenon? Response: Despite the 21 years to life imprisonment for pipeline vandalism [1; Section 2 of the Petroleum Production and Distribution (Anti-Sabotage) Act Cap]; statistics have shown three main aspects of the vandalism; that must be addressed if any meaningful sustainable gain against pipeline vandalism can be addressed. One, Nigeria is losing well over 300,000 barrels per day (BPD) as a result of crude oil pipeline vandalism; [2]. Two, this translates into billions of dollars in losses [3, 4]. Where is this money? Which “class” of Nigerians are involved in this act? Also, pipeline vandalism occurs at remote locations because of the nature of the criminality [5]. In the global fight against the COVID-19, several of our liberties were suspended. This includes the freedom of movement and association. The pandemics, therefore, offer a unique opportunity to study the effect of movement restriction on pipeline vandalism. It is logical to assume a relationship between access to pipelines and physical vandalism (which involves the destruction of pipes materially). This type of destruction is not remote; it implies access. Access to the pipeline and the opportunity; are two aspects of the equation that were removed during the lockdown. “Access” involves proximity to the pipeline during which time these structures are destroyed or compromised to gain the fluid within. Legally, at this point, the miscreant is termed a vandal. “Opportunity“, on the other hand, entails a longer time duration to enable the fluid to be scooped/removed and carted away by the (Vandals, now termed ) thieves.Verification of this model, during “peacetime” is somewhat limited in a democracy. The constitution and economic considerations would not permit. The pipeline passes through a large expanse of land and to effectively lock down the route and passes would be practically impossible. However, during the pandemic, many of these considerations (economically, politically, and constitutionally) were removed, therefore the condition (for vandalism) theoretically diminished. In this light, the pandemic and consequent lockdown could be seen as an experiment. Therefore, the study would reveal the roles “opportunity” and “access to pipeline” play in vandalism. The Researchers could test if a relationship exists between observed variables and their underlying latent constructs. To accomplish this, the researchers use empirical research, to postulate the relationship pattern and test it statistically. Why is it relevant? This paper examines the nexus between oil pipeline vandalism and public accessibility in Nigeria. Given the adverse impact of pipeline vandalism as exemplified in loss of life, economic losses, environmental degradation, and pipeline explosions, the paper submits that an evaluation of the impact of anti-pandemic restrictions on the phenomenon is very relevant as pipeline vandalism poses a danger for economic wellbeing and national security. References: Edward Ohwofasa Okumagba .Oil and Gas Pipeline \"Vandalism\" in Nigeria: Analysing Alternative Options beyond the Traditional Legal Approach. International Energy Law Review. 2019 issue 7 p183-190. https://www.researchgate.net/publication/342184010 Okoli, A.C. The political ecology of the Niger Delta crisis and the prospects of lasting peace in the post amnesty period \"Global Journal of Human Social Science, 2013, 13 (3:1.0), pp.38-46. Michael Eboh 2021. “Cost of pipeline vandalization, oil theft hit N159bn in 1 year”. Vanguard. August 1, 2021. https://www.vanguardngr.com/2020/10/cost-of-pipeline-vandalisation-oil-theft-hit-n159bn-in-1-year/ O. O Udofia; O. F Joel. Pipeline Vandalism in Nigeria: Recommended Best Practice of Checking the Menace. Paper presented at the Nigeria Annual International Conference and Exhibition, Lagos, Nigeria, August 2012. Paper Number: SPE-162980-MS, https://doi.org/10.2118/162980-MS, Published: August 06, 2012 Ahmed Tukur Umar & Moh’d Shahwahid Hajj Othman | Miao Wang (Reviewing Editor) (2017) Causes and consequences of crude oil pipeline vandalism in the Niger Delta region of Nigeria: A confirmatory factor analysis approach, Cogent Economics & Finance, 5:1, DOI: 10.1080/23322039.2017.1353199 1.3. The significance of the paper for future research (i.e., how can this data note help government authorities deal with pipeline vandalism?) Response: The government would be better informed and thus, can make better decisions to checkmate this vice. Section: Methods 2.1. “These monthly reports are available for free download by the public from the NNPC website link (NNPC; https://www.nnpcgroup.com)” To facilitate reproducibility, I kindly ask the authors to provide a more precise URL or path description to derive this dataset. Thank you. Response: Under the land use decree, the oil wealth of the country (Nigeria) resides with the federal government. Most aspect is controlled by the Nigeria National Petroleum Cooperation (NNPC) or its subsidiaries. The NNPC publishes the “NNPC Monthly Financial Operations Report” which can be accessed by clicking “NNPC Business” and selecting “Business Information”, then “Monthly Performance Data” on their website (https://www.nnpcgroup.com) or through (https://www.nnpcgroup.com/NNPC-Business/Business-Information/Pages/Monthly-Performance-Data.aspx) 2.2. “The data were grouped into four groups, namely: [...]” I strongly suggest motivating those subdivisions in detail. In particular, based on what criteria and purpose were these groups formed? Response: The data used were from January 2015 to January 2021. The names of each group are self-explanatory; namely: Historical data, Prior-covid-19,Covid-19 lockdown, And post-COVID- 19 lockdown. The data were grouped based on dates. “Historic data” span from January 2015 to July 2019, these data represent pipeline vandalism data completely without COVID-19 influences. These data were collected before the outbreak, thus as there was no “knowledge” of COVID-19 during the time represented by this group, it could be assumed that COVID-19 did not influence the data collected on pipeline vandalism during this time. These data can therefore be used as a “baseline”; such as “norm”. Groups 2-4 were arbitrarily set within a duration of 6-months. Pipeline vandalism during the time frame represented by groups 2-4 could be imparted by COVID-19: These groups can therefore be compared with “historical data” of the same time in the past from the historical data group, (group1) to determine if COVID-19 had in any way influenced the outcome (the number of pipeline vandalism during the stated time). To minimize/ remove seasonal variation due to the weather (wet and dry season) data were compared only with data from the corresponding seasonal frame. This is logic, data of pipeline vandalism in the summer should be compared against summer data; winter against winter in the colder zones; similarly, data of pipeline vandalism in the rainy season against data of pipeline vandalism in another rainy season in the tropic. Group 1, January 2015 to July 2019, represents a “norm”. However, on closer inspection of figure1, an alteration would be observed about June 2016, this would imply an event occurred which altered the normal sequence. For this reason, literature and newspapers were consulted to determine which event occurred about that time that led to such a drastic change in the norm. The implementation of a different security protocol was found to have occurred in May 2016. Thus, the historical data were further sub-grouped into sub-groups A, B, C, and D, representing 1-year before deployment, the year of deployment, 1-year after deployment, and 2-years after deployment, respectively. For an in-depth explanation, kindly view the Dataverse, Data for Effects of COVID-19 on pipeline vandalism in Nigeria, West Africa, (https://dataverse.harvard.edu/dataset.xhtml?persistentId=doi:10.7910/DVN/8X5KKB) published in Dataverse for COVID-19, social distancing, and pipeline vandalism in Nigeria (https://dataverse.harvard.edu/dataverse/Nneka). 2.3. “May 2016, incorporation and deployment of sophisticated weapons, use of satellite images and geographical information system (GIS) into the security apparatus to ensure vandalism is contained, the setting up of a pipeline security force to stamp out the menace, and the formation of the Trans-National Organized Crime (TNOC) with regional allies to fight against the proliferation of Small Arms and Light Weapons. 4”. Reference 4 refers to a web page called “Oil and Gas 360.” Therefore, I kindly ask if it is possible to provide a more specific URL or refer to an academic source. Response: It is not an academic paper; it is an EVENT reported in the news; hence a newspaper is an appropriate source for this information. The reference is: Levinus Nwabughigu, C. (2016, May 28). Buhari’s milestones in one year – Presidency. Vanguard: https://www.vanguardngr.com/2016/05/buharis-milestones-in-one-year-presidency/ 2.4. “In Nigeria, the pre-lockdown commenced from 28th February – 29th March 2020, and was 31 days in duration. The lockdown, in total 35 days; was from 30th March to 3rd May 2020, and easing up of 73 days, 5th May – 15th July 2020.” This information needs a reference. Response: Ibrahim RL, Ajide KB, Olatunde Julius O. Easing of lockdown measures in Nigeria: Implications for the healthcare system. Health Policy Technol. 2020;9(4):399-404. doi:10.1016/j.hlpt.2020.09.004 and Dan-Nwafor, Chioma et al. “Nigeria's public health response to the COVID-19 pandemic: January to May 2020.” Journal of global health vol. 10,2 (2020): 020399. doi:10.7189/jogh.10.020399 2.5. “The May 2016 event (from a cursory glance of Figure 1) had a great impact on cases of pipeline vandalism.” Figure 1 shows that the decreasing trend has occurred since July 2016. Therefore, it is necessary to argue the causal nature of the association made and discuss the presence of a time-series lag. Response: A “lag” is a fixed amount of time. A lag plot is a special case of x, y plot; wherein a set of observations is plotted lagged against a second set of observations. It is our view that adequate information on the number of cases of pipeline vandalism by time (month/year) is conveyed by figure 1. The possible causal nature/ the association of other events with cases of vandalism are shown in the graph. Yes, it can be argued that an association exists between the decreasing trend after the deployment and the deployment. 2.6. “In all statistical analysis in this project, an alpha = 0.05 as the significance threshold was set, and a null and an alternative hypothesis were established. This means that the null hypothesis would be rejected if the p-value is less than or equal to 0.05 and the alternative hypothesis would be accepted.” Using a simple threshold for significance analysis is misleading (Amrhein et al. (20171), Greenland et al. (20162)). P-values should be used - at best - as a graded measure of the strength of evidence against the null hypothesis. Therefore it is necessary to report the P-values in full (if this is not possible, it is advisable to provide an additional file). Furthermore, P-values are unsuitable for measuring the intensity of a phenomenon (e.g., very weak but statistically significant phenomena can occur (Schober et al. (20183))). Therefore I suggest introducing quantifiers into the analysis (e.g., percentage differences). Response: In recent times, some scholars have challenged the use of a threshold to declare the statistical significance of the p-value, [1]. Two main arguments are, one; research data contain more meaning than is summarized in a P-value and its statistical significance, and two; the concepts are frequently misunderstood and consequently inappropriately interpreted. The abolishment of p-values has been echoed in such an article as “Should statistical significance be retired? “ [DOI: 10.1007/s00393-020-00835-x, download at https://pubmed.ncbi.nlm.nih.gov/32621162/ ]. We, however, do not wish to be drawn into that argument.In line with best practice for transparency in data analysis, our research hypotheses were clearly articulated; they are; namely: Null hypothesis: There is no significant difference between the mean case of pipeline vandalism incidences prior, during, and post COVID-19 lockdown. Alternative hypothesis: There is a significant difference between the mean cases of pipeline vandalism incidences prior, during, and post COVID-19. Traditionally, researchers examine such differences between groups using t-test, Anova [2,3,4]. We used ANOVA; however, in the second version, percentage differences shall be added as quantifiers into the analysis. References: Andrade, Chittaranjan. “The P-Value and Statistical Significance: Misunderstandings, Explanations, Challenges, and Alternatives.” Indian journal of psychological medicine vol. 41,3 (2019): 210-215. doi:10.4103/IJPSYM.IJPSYM_193_19. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6532382/ http://mason.gmu.edu/~afinn/html/teaching/courses/f03_comm250/fbk_chapters/13.pdf https://www.sciencedirect.com/science/article/pii/S1877042813029686/pdf?md5=2ee7f259710d2a331e4a54e73e979d69&pid=1-s2.0-S1877042813029686-main.pdf https://www.arcjournals.org/pdfs/ijsimr/v3-i12/10.pdf 2.7. “The sub-grouped data were also subjected to an ANOVA, and a time series analysis (after the data were smoothened by moving average).” ANOVA analysis requires the verification of a certain number of assumptions, as explained here. Therefore, how the latter has been verified must be described in detail. Moreover, it is necessary to specify the type of ANOVA adopted. Finally, the amplitude of the moving average must be specified. Response: The assumptions in ANOVA are concerned with the population distribution. These three assumptions are: Homoscedasticity of the dependent variable (equality of variances among group Dependent variable is normally distributed within each group Each observation in the sample is independent of all others The type of ANOVA: In this investigation, one-way ANOVA was used. A one-way ANOVA is a statistical test used to determine whether or not there is a significant difference between the means of three or more independent groups. The assumptions in the one-way ANOVA are: Normality: that each sample is taken from a normally distributed population. Sample independence: that each sample has been drawn independently of the other samples. Variance Equality: that the variance of data in the different groups should be the same. The verification of the assumptions used in the ANOVA analysis. Normality can check visually or, by a histography. Variance can be checked with a boxplot. For the third assumption, there is no formal test to verify that the observations in each group are independent. Finally, the amplitude of the moving average must be specified. The amplitude of the moving average shall be elaborated in the second version of the article 2.8. A time-series analysis requires the verification of the absence/presence of previous trends. Therefore, I suggest adding this essential control. Response: This shall be added in the second version of our paper. Thank you for your observation. 2.9. “Pipeline vandalism takes place in remote locations on isolated, rural roads and footpaths; not readily accessible during adverse weather conditions.” Since this sentence justifies a fundamental assumption, I suggest motivating it with a source. Response: Olu-Adeyemi (2020). The Political Ecology of Oil Pipeline Vandalism in Nigeria Lanre. International Journal of Research and Innovation in Social Science (IJRISS) |Volume IV, Issue V, May 2020|ISSN 2454-6186 and Okoli, Al Chukwuma (2019). Oil Pipeline vandalism In the Niger Delta. Accord: Conflict Trendsop. Cit: https://www.accord.org.za/conflict-trends/oil-pipeline-vandalism-in-the-niger-delta/ Minor comments: m1) Section: Method. “The onset of COVID-19 in December 2019 and the declaration of COVID-19, on 30th January 2020, as a Public Health Emergency of International Concern by WHO (World Health Organization), and the upgrade to a pandemic by the 11th of March 2020.” This sentence needs a reference. I can suggest this one here. Sohrabi C, Alsafi Z, O'Neill N, et al. World Health Organization declares global emergency: A review of the 2019 novel coronavirus (COVID-19) [published correction appears in Int J Surg. 2020 May;77:217]. Int J Surg. 2020;76:71-76. doi:10.1016/j.ijsu.2020.02.034 and Cucinotta D, Vanelli M. WHO Declares COVID-19 a Pandemic. Acta Biomed. 2020 Mar 19;91(1):157-160. doi: 10.23750/abm.v91i1.9397. PMID: 32191675; PMCID: PMC7569573. Response: Thank you, we will include it. m2) Section: Method. “During the lockdown, no activists, radicals, and militants made any political statement; [...]” It would be appropriate to justify this sentence with a source. Response: As noted in response to your question in 2.3 above. Everything is not an academic paper. When activists, militants strike a pipeline installation it is reported in the News. This implies an absence of reports in the absence of a strike. Overall, thank you for your observations."
}
]
},
{
"id": "115417",
"date": "11 Jan 2022",
"name": "Nima Khakzad",
"expertise": [
"Reviewer Expertise Safety Engineering",
"Cascading effects"
],
"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 have assessed the influence of satellite/GIS equipment and the COVID-19 lockdown on the number of pipeline vandalism events in Nigeria. In general, the employed technique is oversimplified, and the research outcomes are too obvious to warrant a novel/innovative study. From a methodological perspective, regarding the deployment of satellite/GIS equipment and restriction of social activities due to the COVID-19 lockdown as two independent variables and the number of pipeline vandalism events as the only dependent variable, the authors should have employed a multi-variable correlation analysis rather than two separate single-variable correlation analysis, at least from the onset of the lockdown onwards.\nFurthermore, the results of the study are too obvious to justify the necessity of the research. That the deployment of monitoring measures and the COVID-19 lockdown have had a negative impact on the number of the vandalism events was predictable even with a glimpse on the raw data in Figure 1.\nBelow are some more comments:\nIn the abstract, the data is said to have been assessed in 5 groups, but only 4 groups have been named. Please correct this.\n\nUnder “other assumptions made”, 2 groups of people are said to be involved in pipeline vandalism, but three groups are named. Please correct this.\n\nIn the abstract, it should be pointed out, as one of the main outcomes of the study, why the number of pipeline vandalisms rose again in 2017 despite all the security and satellite measures.\n\nUse of acronyms/abbreviations in the Keywords is not recommended. (NNPC in this case). Besides, the keywords “pipeline” and ‘pipes” are too close to be considered two different keywords.\n\nThe vertical lines marked on Figure 1 need to be in different colors with a legend provided to decode the colors. In the current form, except the fist line which denotes the deployment of satellite/GIS equipment, the other lines are not clear to mark which significant event/date.\n\nIs the rationale for creating the dataset(s) clearly described? Yes\n\nAre the protocols appropriate and is the work technically sound? No\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/10-604
|
https://f1000research.com/articles/11-1535/v1
|
19 Dec 22
|
{
"type": "Research Article",
"title": "Cationic biopolymer decorated Asiatic Acid and Centella asiatica extract incorporated liposomes for treating early-stage Alzheimer’s disease: An In-vitro and In-vivo investigation",
"authors": [
"Akhilesh Dubey",
"Namdev Dhas",
"Anup Naha",
"Usha Rani",
"Ravi GS",
"Amitha Shetty",
"Chaithra R Shetty",
"Srinivas Hebbar",
"Akhilesh Dubey",
"Namdev Dhas",
"Anup Naha",
"Usha Rani",
"Ravi GS",
"Amitha Shetty",
"Chaithra R Shetty"
],
"abstract": "Background: Asiatic acid (AA) is a naturally occurring triterpenoid derivative of Centella asiatica (CA) with neuroprotective effect. The study aimed to design an ideal oral drug delivery system to treat Alzheimer's disease (AD) and develop chitosan-embedded liposomes comprising an extract of CA (CLCAE) and compare them with the chitosan-coated liposomes of asiatic acid (CLAA) for oral delivery to treat the initial phases of AD. Methods: The solvent evaporation technique was used to develop CLCAE and CLAA, optimised with the experiment's design, and was further evaluated. Results: Nuclear magnetic resonance (NMR) studies confirmed coating with chitosan. Transmission electron microscopy (TEM) and atomic force microscopy (AFM) indicated the successful formation of CLCAE and CLAA. Differential scanning colorimetry (DSC) confirmed the drug-phospholipid complex. Furthermore, the rate of in vitro release of CLCAE and CLAA was found to be 69.43±0.3 % and 85.3±0.3 %, respectively, in 24 h. Ex vivo permeation of CLCAE and CLAA was found to be 48±0.3 % and 78±0.3 %, respectively. In the Alcl3-induced AD model in rats, disease progression was confirmed by Y-maze, the preliminary histopathology evaluation showed significantly higher efficacy of the prepared liposomes (CLCAE and CLAA) compared to the Centella asiatica extract (CAE) and they were found to have equivalent efficacy to the standard drug (rivastigmine tartrate). The considerable increase in pharmacodynamic parameters in terms of neuronal count in the CLAA group indicated the protective role against Alcl3 toxicity and was also confirmed by assessing acetylcholine (Ach) levels. The pharmacokinetic study, such as Cmax, Tmax, and area under curve (AUC) parameters, proved an increase in AA bioavailability in the form of CLAA compared to the pure AA and CLCAE forms. Conclusion: The preclinical study suggested that CLAA was found to have better stability and an ideal oral drug delivery system to treat AD.",
"keywords": [
"Asiatic acid",
"Centella asiatica extract",
"Neuroprotective",
"Chitosan-coated liposome",
"Alzheimer's disease",
"Oral bioavailability"
],
"content": "Introduction\n\nThe most widespread form of dementia throughout the world is Alzheimer’s Disease (AD), which possibly involves environmental and biological factors leading to chronic, progressive neurodegeneration.1 The ubiquity rate increases by approximately 1% to 40% at 60 to 90 years.2 In the progressive stage of AD, amyloid plaques and tangled bundles of proteins are responsible for blocking neurotransmission signals, resulting in short-term memory loss, anterograde amnesia, frustration, and mood swings associated with neuropsychiatric problems.3,4 Therefore, diagnosis and early stages of treatment, i.e., long-term and concomitant medication are essentially required to manage AD effectively. Currently, there is no specific medication available to cure AD completely. However, some studies have reported that progression can be delayed by treating patients with cholinesterase inhibitors. Cholinesterase inhibitors were shown to prevent the breakdown of Ach neurotransmitters responsible for memory and thinking.5 The translation of natural medicinal treasures into a suitable form for preventing or curing a particular disease is emerging in various research domains.6 The use of phytoconstituents in treating diseases is more promising due to their clinical potential, with a better safety margin and cost-effectiveness than synthetic alternatives.7 Centella asiatica (CA) plants have the highest concentration of triterpene glycosides. Asiatic acid (AA) has acquired prominence among triterpenoids due to its various pharmacological activities against a variety of illnesses.8 AA (Figure 1) has been discovered as the most active chemical capable of rejuvenating neurons. It safeguards against the hippocampus' neurogenesis declining and memory deficits.9–11\n\nThe oral route is the most suitable mode for long-term medication with its ease of administration, coupled with the highest degree of patient compliance and reasonable treatment persistence.12 However, most phytoconstituents possess poor aqueous solubility, low permeability, and diffusibility, which affect their bioavailability. Similarly, poor solubility of AA results in poor absorption, leading to compromised bioavailability, thereby limiting its clinical use. The efficacy of any formulation is determined by the amount to which active ingredients are delivered to the target location at the required therapeutic concentration and exposure period.13,14 Liposomes are nano-vesicular systems for targeted drug delivery that protect the drug from the external environment. This transport system can bypass the drug degradation in the liver, thus enhancing bioavailability.15 Conventional liposomes for oral distribution are unsuitable because they degrade rapidly in the environment of gastric acid and other metabolic enzymes found in the gastrointestinal (GIT) system. Conventional liposomes can be modified by including polymers, macromolecules, polysaccharides, antibodies, or aptamers to advance the brain's targeted delivery and blood circulation time.16 The chitosan-coated liposome is one such modification that improves oral drug delivery. Chitosan is a biodegradable polymer that contains d-β (1→4)-linked N-acetyl-D-glucosamine (A) and D-glucosamine (D) sugar units. Due to its ionic charged interlinkage between a negative charge phospholipid and a positive charge amino group, chitosan strengthens stability and prevents drug degradation from the lipid bilayer by creating a long-lasting biocompatible coating on the lipid membrane. Furthermore, the mucoadhesive characteristics of chitosan result in a prolonged residence period well at the location of absorption and extended release of the drug.17 The oral efficacy of AA can be altered significantly by incorporating it into chitosan-coated liposomes. It is a promising approach to halt the progress of the disease in the early stages because of its convenient mode of administration with resistance to enzymatic destruction and exhibits sustained drug release.18,19\n\nTherefore, the work is intended to fabricate and compare chitosan-coated liposomes of Centella asiatica extract (CLCAE) and chitosan-coated liposomes of asiatic acid (CLAA) to promote the oral absorption and bioavailability in the treatment of the initial stages of AD.\n\n\nMethods\n\n97% pure asiatic acid and 75–85% deacetylated chitosan of 50kDa were acquired from Sigma-Aldrich, USA. Centella asiatica extract was prepared using the soxhlation method in the college laboratory. “Phospholipon® 90 G” was obtained as ex-gratis from Lipoid®, Germany. Rivastigmine was acquired from Yarrow Chem Products. Cholesterol, dichloromethane, dimethyl sulphoxide (DMSO), and methanol were purchased from Himedia Laboratory Private Limited. Chemicals & reagents like orthophsosphoric acid and acetonitrile were analytical or HPLC grade and did not require any extra purification.\n\nThe NGSM Institute of Pharmaceutical Sciences' Institutional Animal Ethics Committee (IAEC) approved the purchase of adult Wistar rats (both male and female) weighing approximately 200–250 g, from the Nitte University-affiliated centre for animal research and experimentation (NUCARE) (Approval No -NGSMIPS/IAEC/MARCH-2018/88). In accordance with committee for the purpose of control and supervision experiments on animals (CPCSEA) rules, experiments were conducted. Six groups of six animals each were maintained in a cage, with the optimum conditions of 25°C, 50% RH, and 12-hour light/dark intervals with regular feedings including water and pellets food (Krishna valley Aggrotech, Sangli, Maharashtra, India).\n\nTo maximize phytoconstituents, the CA plant was harvested early in the morning from a fertilized area during the rainy season.20 The plant was authenticated, followed by preliminary evaluations. The CA plant was dried at 45°C for two weeks, powdered, and subjected to soxhlet extraction with the solvent methanol. The selection of the solvent was based on the phytochemical test. The CA extract was filtered, dried, and stored airtight for further studies. The amount of phytoconstituents, mainly AA, considered a standard drug for further investigations in the extract, was estimated using the RP-HPLC method.\n\nThe liposomes are prepared according to the modified solvent evaporation method.21 AA, SPC (soy phosphatidylcholine) and cholesterol were mixed in various ratios with 25 mL of dichloromethane reagent in 100 mL round bottom flask. To acquire the film, it was attached to a rotary flash evaporator with a set 60 rpm speed at 60°C. Phosphate buffer pH 6.5 was taken to hydrate the film at ambient temperature.22\n\nDesign expert software was utilised to explore the outcome of various parameters to get an optimised formulation. The effect of parameters, such as the molar ratio of drug: lipid (X1) and the ratio of SPC: cholesterol (X2) as independent variables, on vesicle size (Y1) and entrapment efficiency (Y2) as dependent variables was investigated at three levels: low, middle and high concentration representing -1, 0 and +1 respectively.23,24 The software Design Expert version 11.0.3.0;64-bits stat- Ease, Minneapolis; USA (RRID:SCR_002427) prepared and evaluated for the response suggested nine formulation combinations. The obtained results were analysed and validated using an analysis of variance (ANOVA)(RRID:SCR_002427) The following quadratic equation was taken to assess the implied model:\n\nWhere “Y” is the sum of calculated responses from each factor level, “b0” is an intercept. The regression coefficients “b1” to “b22” are calculated from the experimental values of “Y,” and “X1” and “X2” are the implied levels of independent variables. Table 1 displays experimentally obtained values of independent variables.\n\n1H-NMR spectrometry is employed to quantify the drug's encapsulation in a bilayer lipid membrane. The 1H-NMR spectra of pure AA, SPC, and the optimised liposome formulation were taken and analysed for the carbon-hydrogen framework of every single component.25 A specific quantity of samples was liquified in DMSO, filled into NMR tubes, and analysed by a 400 MHz Fourier transform nuclear magnetic resonance (FTNMR) spectrophotometer (Bruker Ascend, Rheinstatten, Germany).\n\nThe modified ionotropic gelation was used to design CLCAE and CLAA.26 In 0.5% w/v acetic acid, chitosan was dispersed. The mixture was maintained at room temperature overnight. For one hour, the mixture was treated with dropwise optimised liposomal suspension while being continuously magnetically stirred at room temperature. Liposomes were left undisturbed for 3–4 hours to swell. An ultra-probe sonicator was used to sonicate liposomal suspension for 30 minutes to produce chitosan-coated liposomes. The prepared formulation was kept at 5°C in an airtight container for further studies.27\n\nSize of the vesicles, poly dispersity index (PDI), and zeta (ζ) potential\n\nThe formed vesicle average size, PDI, and ζ-potential of optimised CLCAE and CLAA were observed by the Malvern zeta sizer, which operates based on the dynamic light scattering principles. Prior to analysis, sample dilution was made in the ratio of 1:10 with the ion-free water. To assess the physicochemical parameters and stability of the synthesised liposomes, the study was triple-checked at 25°C.\n\n% Entrapment efficiency (EE)\n\nA quantitative method was used to determine the % EE of the optimised CLCAE and CLAA.28 A white pellet was formed by centrifuging the sample at 20,000 rpm for one hour at 4°C in a refrigerated centrifuge. The unentrapped drug was examined by separating the supernatant. The sample remaining in the basal portion of the tube was mixed with 0.1N 500 μl of sodium hydroxide and meticulously vortexed for three minutes. The Triton X-100 was added 5mL to make a clear colloidal mixture and further centrifuged for two more minutes to separate the drug from the enveloped vesicles.\n\nThe % of the entrapped drug was determined using RP-HPLC using the following formula,\n\nHebbar et al. developed and validated the method of RP-HPLC technique for AA as per ICH harmonised tripartite guideline.29 The stationary phase was column C-18 with 250 mm x 4.6mm x 5μ (Phenomenex Luna Omega) and the solvent system included 0.1% orthophosphoric acid and acetonitrile with 1 mL/min flow. Photo diode array was used as a detector at a wavelength of 210 nm. The calibration curve was plotted using a stock AA solution. The method's limit of detection and limit of quantitation were found to be 0.784507 μg/mL, and 2.615 μg/mL respectively. The regression equation was y=3790.1x-3001.9, 9.69.6±0.22 minutes was the retention time for the AA and 0.9987 was the correlation coefficient (r2).\n\nThe AA content in both liposomal formulations (CLCAE and CLAA) was determined through the RP-HPLC method as mentioned above. The formulations (1 mL each) were dispersed separately in 10 mL of methanol. The samples were membrane filtered and diluted appropriately to estimate the drug content.\n\nMeasurement of turbidity determines the stability of CLCAE and CLAA as per the study reported by Chang-Moon Lee et al.30 CLCAE, CLAA, and uncoated liposomes were added to SGF. The pH 1.2 SGF was made using 0.2% w/v sodium chloride, 0.7% v/v of hydrochloric acid, and 0.32% w/v pepsin. Turbidity and chitosan concentrations are interrelated; the more the turbidity, the more probable it is that chitosan will be lost from the vesicles. The turbidity of the liposomes was measured using a Digital Nephelo Turbidity Metre, and the results were represented in nephelometric turbidity units (NTU).31\n\nThe specific changeover temperature difference of the samples such as AA, SPC, its mixture and formulation CLAA were measured through DSC (Q20, TA Instruments USA).32 The samples were exposed to the temperature 0 to 400°C at a heating rate of 10°C/min. The universal software version 4.5A, TA instruments USA was taken to quantify peak transition temperature.\n\nThe morphological characteristics such as the shape and appearance of CLCAE and CLAA were determined through TEM (version JEM-100s, JOEL, Japan).33 The samples were diluted in the ratio 1:20 with the ion-free water before the analysis and further sonicated for 3 min. The sample droplet was individually deposited onto a metal plate made up of copper that had been coated with carbon to create a thin film. One drop of colouring pigment i.e., 2% w/w ammonium molybdate with 2% w/v ammonium acetate of pH 6.8 was added to the film. It was examined under a transverse electron microscope and compared both the sample surface morphology.\n\nThe three-dimensional surface structure analysis of the formulation CLCAE and CLAA were carried out through AFM (Innova SPM, USA).34 Each sample was placed separately as a smear and observed through the range of resonance frequency 267-329kHz with the scanning speed 1.2Hz of AFM tips.\n\nThe % drug release from CLCAE and CLAA were measured through Franz diffusion cell using sigma dialysis membrane and the release rate was compared with AA and CAE. 1 mL sample (100 mg/mL) was kept on one side, while the other part of the membrane was filled with the acidic buffer pH 1.2 (200 mL) dissolution media for two hours. It was then replaced with phosphate buffer pH 7.2 (2–24 hours) with 0.25% w/v sodium lauryl sulphate as two separate media to simulate the stomach and intestinal conditions. The media were sustained at the temperature of 37°C on a magnetic stirrer. A 5 mL sample was withdrawn with the specific intervals and the condition of the sink was kept constant by refilling the same volume of media. Samples were analysed and compared using RP-HPLC at 210 nm. To assess the drug release strategy from the optimised CLAA, the data were fitted to various in vitro kinetic release models like Korsmeyer-Peppas, Higuchi, Zero and First Order models.35,36\n\nThe prepared formulations' and AA's abilities to neutralise free radicals were determined in vitro and compared with standard ascorbic acid. In this method, free radical DPPH (2,2-diphenyl-1-picrylhydrazyl) was selected as described by Jamuna S. et al.37 1.5 mL of 0.1mM free radial DPPH was treated with 3.5mL of 10 to 50 μg/mL concentrated methanolic solution of AA, CAE, optimised CLCAE, and CLAA.38 The absorbance of all the samples was measured at 518 nm through ELISA plate reader (version AM-2100, USA). The percentage of DPPH inhibition was measured through the formula.\n\nThe drug intestinal permeation studies of both the optimised formulations were determined using non-everted gut sac procedure.39,40 The rats were sacrificed and small intestine was treated with oxygenated saline. The intestine was split into 30.5 mm-diameter sacs that were 8 cm long and loaded with optimised CLCAE and CLAA (~100 mg AA). The thread was used to bind the sac's ends and located in a conical flask filled with ringer’s solution of pH 9 (10 ml). 37°C temperature was kept constant in a water bath shaker at 75 rpm with 5% CO2 aeration. Every 20 min, for a maximum of 8 h, the samples were removed and substituted with the fresh media. RP-HPLC was used to analyse the screened sample.\n\nExperimental design\n\nThe animal experiments were carried out in the month of January (17/01/2019). The treatment (once a day, p.o) was administered for 89 days as described below:\n\n1. Normal control: 0.9 % w/v NaCl, (5 ml/kg)\n\n2. Disease control: AlCl3 (50 mg/kg)\n\n3. Positive control: AlCl3 (50 mg/kg) + Rivastigmine (1 mg/kg)\n\n4. AlCl3 (50 mg/kg) + CAE (5 g/kg)\n\n5. AlCl3 (50 mg/kg) + CLCAE (100 mg/kg)\n\n6. AlCl3 (50 mg/kg) + CLAA (100 mg/kg)\n\nThe behavioural assessment was performed using the Y-maze to evaluate disease induction on the 45th day and 90th day, followed by a histopathology study. The experiments were concluded on the 95th day (21/04/2019) from the first day of the experiment.41–44\n\nAluminium ions are a potential neurotoxic agent. This trivalent cation binds to IRP (iron regulatory protein) and stimulates AβPP as well as ferritin. APP's inappropriate overexpression will result in more Aβ being produced (which is resistant to protease enzyme because Al ion associated), accumulates mainly in the hippocampus region. By promoting tau phosphorylation and iron-induced lipid peroxidation, Al ions contribute to neurodegenerative processes. A change in the concentration of free iron ions, the production of free radicals, and the aberrant expression of ferritin all contributed to oxidative damage and membrane lipid peroxidation. In AD, these occurrences ultimately result in neuronal death.45\n\nThe Y-maze model was used to determine the spatial working memory and assess rodents’ continuous altering behaviour. In this method, the animals were involved in a suitable search operation using food as a reward. The animals were trained prior to the commencement of the experiment.46 The Y-maze consisted of three horizontal arms that were allied at an angle of 120°. The maze arms had walls of 40 cm in length, 3 cm in width, and 12 cm in height. The three arms were labelled as the start arm in which the animal started to explore (A), a reward arm containing food stimuli (B), and another random arm (C). The maze was made up of dark polyvinyl plastic that was opaque. For the test, the animals were put in the start arm and given free rein to explore the maze. The sequence of each arm and entry was recorded for 8 min. The arm entry was tabulated; only alternated arm records such as ABA, BCB, CBC, and so on—rather than repeated arm entries were taken into consideration during the trial. The arm entries and the alternations were recorded, and % alternations were measured.\n\nThe locomotor activity of the animals was estimated with the help of the arm entries taken place.47 All experiments were carried out under standard laboratory conditions.\n\nThe rats were euthanized on the 90th day. The brain was perfused using ice-cold normal saline and then dissected to isolate the frontal cortex and hippocampus. Hippocampus was sectioned, and tissue processing was done. The tissue was stained with 0.1 % crystal violet stain and observed under the microscope (Zeiss Primo Star Digital, Germany). Photographs of the CA1 and CA3 areas of the hippocampus were taken, and the number of normal healthy neurons out of 100 neurons was counted with the help of image J software (version 1.53t).48\n\nAcetylcholinesterase (AChE) is primarily a major enzyme in the cholinergic system. AchE was estimated by the Ellman method.49 To the rat brain tissue homogenate (100 μl), 0.1 M phosphate buffer pH 8 (650 μl) and DTNB (Ellman reagent) (0.1 mL) were blended after being added. The mixture was treated with 0.1 mL acetylthiocholine iodide, and a UV-visible spectrophotometer at 412 nm, the absorbance was determined (UV-1800, Shimadzu, Japan). The brain tissue homogenate was substituted with 100 μL of purified water to make the blank.\n\nEstimation of AA in rat serum\n\nThe sum of drug availability after oral intake of AA, CLCAE, and CLAA was performed on three groups of 150-200g wistar rats (n=6). Prior to the procedure, the animals underwent an overnight fast with unlimited access to water. Group I was treated with a single dose of AA (100 mg/kg,p.o.) whereas group II, and III animals received single dose of optimised CLCAE and CLAA (~100 mg/k.g AA, p.o.), respectively. After the dosing, the animals were partially anesthetised using either, from the retro-orbital plexus 1 mL of blood samples were collected in centrifuge tubes at a predetermined time of up to 8 h. To isolate the contents, the serum sample was spun up at 3000 rpm for 10 min. The AA was quantified by RP-HPLC.50\n\nExtraction of AA from plasma\n\nInitially, the mixture of 1 mL serum and 5 mL methanol were taken in a volumetric flask at room temperature. The sample was properly mixed before being heated for 30 min at 55°C. After that, a 10 mL flask was made up with methanol and centrifugation for 30 min at 5000 rpm (Remi Elektrotechnik Ltd. India). 20 μL of filtered supernatant was taken for analysis.\n\nPharmacokinetic measurements\n\nDepends on plasma concentration-time curve parameters, the Cmax and Tmax of CLCAE and CLAA were analysed. The pharmacokinetic parameters were successfully interpreted through PK/PD computer software version 4.1, USA, which includes measurement drug plasma concentration from zero to last measured sample (AUC0 -t) and from zero to infinity (AUC 0-∞) measurement of drug elimination half-life (K1/2el) and elimination rate constant (Kel) clearance (Cl) and volume of distribution (Vd). The relative bioavailability (F) of both the formulations was determined using the following formula.51\n\nThe obtained data of behavioural parameters and pharmacokinetic studies were stated as mean±standard error mean (SEM). Student t-tests and one-way analysis of variance (ANOVA) were used for the statistical analyses. Statistical were regarded for P values <0.05.\n\nThe formulated optimised CLCAE and CLAA underwent a stability investigation for three months at two distinct settings, refrigeration and room temperature (5 ±2°C), (32 ±2°C) at 60 ±2% RH, respectively. Up to three months, each month samples were taken, diluted appropriately with 7.4 pH buffer for determine EE, vesicle size and zeta potential52\n\n\nResults and Discussion\n\nCA plants that were harvested, were primarily investigated for quality criteria. The safety of its consumption was determined by total ash value, insoluble acid ash value, and water-soluble ash value, which were found to be 16±0.23%, 3±0.21%, and 3±0.43%, respectively. CA showed the total moisture content of 7.2 ± 0.62%, signifying better stability. The methanol solvent in the soxhlet extraction process yields maximum phytoconstituents compared to solvents like, chloroform, petroleum ether, n-butanol, ethyl acetate and solvents screened by chemical tests. Methanol's amphiphilic nature allows it to dissolve almost all compounds irrespective of the polarity. The obtained extract was standardised and its terpenoids estimated using the RP-HPLC method. AA was considered a reference compound for subsequent drug quantification; with a retention time of 15.79 min, the compound AA was identified, which was found to have approximately 10% availability in the total extract.\n\nThe pre-formulation study was conducted to improve the solubility of AA. AA is a BCS IV drug that has poor aqueous solubility and permeability. Therefore, organic solvents were used in the initial screening. A clear solution was formed in dichloromethane that also possessed a reduced boiling point (39.6°C) and reduced toxicity (LD50 value of 1.2 g/kg in rat p.o). Therefore, to prepare the formulations, dichloromethane was chosen as a solvent. The drug: SPC ratio was set at 1:5 to 1:15 based on the literature research, and the SPC: cholesterol ratio was set at 70:30–50:50 based on the DoE. A rotary flash evaporator was used to evaporate mixtures that had been liquified in dichloromethane solvent. The film was dried for nearly 45 min at 45°C using a vacuum pump before hydration to remove the organic solvent. Because liposomes are prone to fusion and drug leakage, to enhance its rigidity and stability, cholesterol was added.53,54 Using an ultra-probe sonicator, the resulting multilamellar vesicles (MLVs) were pulverised to reach the required size range of less than 250 nm. Furthermore, an investigation was conducted to study the factors that influence the formulations.55\n\nTable 2 displays the results derived using a three-level multifactorial randomised polynomial equation model for the two dependent factors for the range of formulations according to the study design.56 The obtained values from the experimental trials exhibited a considerable variation in vesicle size (142-277 nm) and % EE (56.08%-82.38%). The model's as well as its parameters' relevance generated, were analysed by ANOVA. The conclusions were made based on polynomial equations (equations 2 and 3). The positive sign before the factors indicated that the response and the factor had a linear relationship, the negative sign, on the other hand, denoted inverse correlation between both.\n\nA and B the implied values for the AA: SPC and SPC: cholesterol ratios, correspondingly. The model developed for vesicle size was significant since it had a p-value of 0.05 and an F-value of 7.87. The difference between the adjusted R-squared value (R2 = 0.6318) and the predicted R-squared value (R2 = 0.5257) was less than 0.2, indicating that the models agreed rather well. As shown in equation 1, the AA: SPC ratio had a prominent effect on vesicle size. The model developed for entrapment efficiency was significant, with a p-value of 0.05 and an F-value of 35.72. The difference between the adjusted (R2 0.8967) and predicted (R2 0.8080) below 0.2 R-squared value indicated good correlation. As shown in equation 2, the AA: SPC ratio and the SPC: cholesterol ratio significantly affected the entrapment efficiency. The prepared liposomes were found to have a vesicle size of 140 to 280 nm and a % entrapment efficiency of 50% to 80%. The optimum concentration was selected based on desirability values (Table 3). The vesicle size of the optimised liposome was 209.8 nm, and the percent entrapment efficiency was 71.2 ±0.03 %.\n\nThe 1H-NMR spectrum of pure AA is in Figure 2A, which shows different types of protons and its allocation corresponds to the type of hydrogen in the full structure of AA. The SPC 1H-NMR spectra are in Figure 2B. Basic chemical displacement values were observed. The chemical shifts of pure AA and optimised liposomes of AA were examined, with the downfield aromatic region (>7) and the upfield aromatic region,4 showing most significant differences. The alkyl side chain of the optimised liposome was observed at δ 2.50, and the N-methyl groups corresponded to the chemical shift at δ 3.81. The alteration in proton signs in the aromatic area clearly demonstrated the establishment of molecular bonds with AA. The molecular interface was indicated by the weak intermolecular interaction between the phospholipid mixture and the phenolic region of AA. This confirms the formation of bilayer vesicles with AA.57\n\nIn order to strengthen the mucoadhesiveness to the negatively charged cell membrane, chitosan was coated on their surface. This enhanced colloidal stability and regulated release.58 It has been discovered that covering the surface of negative charges liposomes with chitosan is made easier by ionic exchanges among both the negative charge lipid group with the positively charged amino group of chitosan.59 Drug leakage from the vesicular structure is also prevented by chitosan coating.60\n\nVesical size, PDI, and zeta (ζ) potential\n\nThe influence of the structural integrity of the liposomes in a media is determined by vesicle size, PDI, and zeta potential.61 When compared to conventional liposomes, chitosan covering increases the width and thickness of the vesicular system. Sonicated optimised CLCAE and CLAA have typical vesicle sizes of 224.4 nm and 209.8 nm, respectively., which signifies the optimum complex formation in which drug molecules tangibly bond with the lipid's polar heads to reduce the negative surface charge and the vesicle size. It plays a role in enhancing AA's absorption efficiency and sustained-release action. Optimised CLCAE and CLAA exhibited PDI values of 0.457 and 0.493, revealing that vesicle size distribution is in a narrow range.62 ζ potential measurement is an essential tool to evaluate the surface electrical charge that indicates the potential stability of a vesicular system. Surface charges ranging from -20 to +20 mV have been found as a feature of ζ potential affinity accompanied by a stronger coagulation process than repellent force.63 The phosphate group has a negative charge in the presence of water. The chitosan coat, on the other hand, contains more cationic polymers adsorbed to the surface of the liposome, resulting in a small positive charge. Optimised formulations CLCAE and CLAA displayed ζ potentials of 22.3 mV and 20.8 mV, respectively. The positive ζ charge of optimised CLAA shows good physical stability that ultimately favours the mucoadhesion property of the cell membrane and penetrates the mucous membrane.\n\n% EE\n\nBy breaking the optimised liposomes using Triton X-100, the % EE was determined. The overall entrapment of AA in optimised CLCAE and CLAA was found to be 51.3 ± 0.03% (n = 3) and 71.2 ± 0.1% (n = 3), respectively. The results of the various studies on liposomal drug delivery and comparison with each formulation were satisfactory. Due to AA's limited water solubility and lipophilic character, EE has a higher propensity to entrap AA in liposome lipid bilayers due to its persistent vesicular shape. After an acid treatment or dialysis method, the unentrapped drug can be removed by centrifugation. To avoid drug loss in this study, the formulation preserved the unentrapped drug.\n\nMethanol was selected as a solvent because AA is insoluble in water. The standard calibration curve was plotted under a 10–50 g/ml linearity range. The regression equation was Y=3790.1x. The correlation coefficient (r2) was 0.999 and the retention time was found to be 9.6±0.22 min. In the optimised CLCAE and CLAA formulations, the AA content was 43 ±0.02% w/w and 68±0.04% w/w, respectively.\n\nIn the oral delivery system, the constancy of liposomes in the gastric fluid is a significant consideration. To confirm the stability of CLCAE and CLAA in comparison to uncoated liposomes, an in vitro stability test was performed. CLCAE and CLAA exhibited turbidity value of 11.2±0.004 NTU and 10.3±0.003 NTU, respectively. In contrast, uncoated liposomes showed 18.5±0.001 NTU, which confirmed the leakage of vesicles. This study indicated the stability of CLCAE and CLAA in SGF. The results of the current parameter indirectly support the better mucoadhesive by the electrostatic interaction of the chitosan-coated formulations.64\n\nDSC is a well-known method for investigating the thermal behaviour to describe complicated solid-state matter.65 Purified AA exhibited a broad endothermic peak at 158.9 °C, which was in line with its melting point, as shown in Figure 3A. At 172.02°C, the SPC exhibited an endothermal peak (Figure 3B), indicating that the physical state shifted from a gel to liquid form. The phospholipid's carbon group could have resulted in a different atom arrangement or crystal modifications. Two peaks were identified for the physical mixture of SPC and AA at 171.96°C and 159.02°C, respectively, matching to the peaks of SPC and AA. Furthermore, the optimised CLAA thermogram revealed a broad endothermal peak at 127.6°C (Figure 3D). The fact that the peak shifted to lower temperatures could be attributable to the drug's improved solubility and lower crystallinity in the formulation form. It could be owing to hydrogen bonding or van der Waals forces combining AA with the tail of phospholipid molecules, resulting in the drug lipid complex.66,67\n\nTEM\n\nTEM images of both optimised CLCAE and CLAA (Figure 4 (A, B)) were well-developed, detached, without any accumulation of vesicles, with particle size in the range of 196 nm and 187 nm, respectively. The obtained particle size of fabricated nanoconstructs can be considered as an ideal particle size for crossing blood brain barrier and bypassing reticulo-endothelial system (RES).68\n\nAFM\n\nThe distinct, well-formed spheres are revealed to be free of any agglomeration or degrading signs shown in the AFM image of optimised CLAA (Figure 5B) and it might be the reason for the improved dissolution profile compared with the pure drug. However, the optimised CLCAE in (Figure 5A) shows a vesicular nanostructure with a small aggregation and non-uniformity; this may be due to the vesicles' leakage, indicating less stability.69\n\nThe rate of drug release from the formulation CLCAE, CLAA, and pure form of AA would be useful to correlate in vivo drug release rate. In around 10 hours, AA exhibited a fast release of 65.34 0.30%. For AA, there was a further rapid decrease in drug release that persisted up to 16 hours. In optimised CLCAE, the sustained-release pattern was found, with CLAA showing 69.43% and 85.3 0.3% release in 24 hours, respectively (Figure 6). The enhanced and sustained drug release from optimised CLAA is caused by the physicochemical alteration and electrostatic interactions of AA with SPC and chitosan. It presumably improved the complex's solubility along with wettability when compared to pure AA.70 Kinetics of drug release of optimised CLAA was examined. Higuchi's plot of the optimised CLAA was linear with an R2 value of 0.972 and verified diffusion regulated drug release based on Fick's law. Korsmeyer-Peppas model showed n value being less than 0.4 (i.e., n = 0.3917), indicating quasi-fickian model. It depicts the drug layer's partial diffusion pattern.71 The Higuchi plot was revealed to be the model that fit the data the best due to diffusion mechanism involved.\n\nThe inhibition percentage of DPPH by AA, optimised CLCAE, and CLAA was compared to the reference ascorbic acid by taking equal concentration. At 50 μg/mL, the percentage of inhibition of DPPH for ascorbic acid, AA, optimised CLCAE, and CLAA, was found to be 90.75 ± 1.45%, 65.4 ±1.2%, 60 ± 1.3%, and 59.84 ± 1.6%, respectively (Figure 7) (n ≥ 3). The results indicated that the pure drug exhibited substantial free radical scavenging activity and was also observed in liposomal formulations. It could be related to the AA's increased capacity to give hydrogen ions and to convert DPPH radicals to hydrazine equivalents.69 Even after interacting it with the chitosan-coated phospholipid layer, AA's free radical scavenging action was intact.\n\nUsing everted and non-everted intestinal sac models, it is possible to quantify the drug reception in the intestinal area.72 Additionally, the non-everted sac models have a number of noteworthy advantages, including lesser volume of the sample and minimum structural damage to the intestine. The transport mechanisms were evaluated, and their relationship to in vivo drug absorption was correlated, using an ex vivo permeation analysis. In this study, the % permeation rate of optimised CLCAE and CLAA was analysed (Figure 8) (n ≥3). The amount of drug permeated throughout 8 hours by optimised CLAA was better (97.9 ± 4.3%) than the optimised CLCAE (93.89 ± 4.03%) and showed a significant penetration rate difference. It most likely happened as a result of the chitosan's bio-adhesive ability. which causes a higher retention period and a prolonged adsorption rate in the mucosal region. The pure AA and CAE suspension demonstrated lesser permeation i.e., 30.90% ± 0.9% and 21.49% ± 0.76%, respectively via intestinal barrier as compared to CLAA and CLCAE. Thus, the results revealed that CAE and AA when incorporated in the nanocarrier may enhance its permeability via intestinal gut lining. Additionally, there was no substantial change in the % permeation between AA and CAE.\n\nThe wistar rat model was chosen for neurological investigations because it is easier to deal with, has a larger brain than the transgenic mice, and is less sensitive to human handling. Rats have relevant gene sequences (AβPP) for AD that are similar to human sequences (96.6%), merely three Aβ sequence differences. The Y-maze model was used to evaluate the effects of formulations on spatial working memory based on two parameters, i.e., the number of arm entries and the percentage alternations.73 The memory retention activity of groups III, IV, V, and VI in terms of the number of arm entries by rats was carried out. Figure 9A shows groups V and VI (dose of ~100 mg/kg AA) exhibited a considerable drop in the sum of arm entries equated to group I. When compared to group I, group II exposed a substantial (p<0.05) increase in the number of arm entries, indicating a disturbance in memory and learning. Group IV (dose of 5 g/kg) showed fewer arm entries than group II. One-way ANOVA was used in the statistical analysis, followed by Bonferroni multiple comparison tests. The samples showed a dose-dependent and significant (p<0.001) rise in the percentage alterations compared to group I. When compared to group I, Group II showed a significant (p<0.001) decrease in percent alterations, which represents a disturbance in memory and learning. Figure 9B shows groups V and VI exhibited a significant (p<0.001) rise in the percent alteration when compared with group I. CAE, CLCAE, and optimised CLAA were found to preserve memory and learning in treated rats even after induction of AD. The optimised CLAA showed more promising results (p<0.05) than the CAE and CLCAE, which could be due to the greater bioavailability of AA.\n\nNote (bottom) Figure 9A: Data represented as mean ± SD. Statistical analysis was performed one way ANOVA followed by Bonferroni multiple comparison tests. ## p< 0.05, ** p< 0.05, b p < 0.01, c< 0.01 when compared to disease control.\n\nNote (bottom) Figure 9B: Data represented as mean ± SD. Statistical analysis was performed one way ANOVA followed by Bonferroni multiple comparison tests. ## p< 0.01, ** p< 0.05, b p < 0.01, c< 0.01 when compared to disease control.\n\nThe processed brains of experimental rats were stained with crystal violet and eosin and examined by optical microscopy (Figure 10). Group I revealed the presence of maximum neurons in cornu ammonis CA1 and CA3 regions and distinguished layers associated with the small blood vessels. The layers such as a plexiform layer, external and inner granular layer, and polymorphic cells were found. Besides this, the dentate gyrus was found to be healthy with a pale, and a round nucleus, and also well-defined nuclear boundary and prominent nucleoli were found.74 Group II exhibited a massive cellular degeneration in the hippocampal region followed by neurofibrillary depletion. Dentate gyrus was found to be damaged darkly (basophilic) stained, with a shrunken and fragmented nucleus. The accumulation of aluminium in these regions leads to the formation of amyloid proteins.75–77 In group III, loss and damage of the neurons were found in the CA3 area. However, not much damage and loss of neurons were found in the CA1 area. In this group, protection over neurodegeneration was observed. Group IV showed significant protection of neurons compared to group II. Group V and group VI showed loss of neurons in the CA1 region and dentate gyrus areas distinct protection compared to group II animals.\n\nThe CA1 and CA3 regions of the hippocampus sector are exposed to AD-type neurofibrillary degeneration.77 The principal reason for a diminution of a neuron is the accumulation of amyloid plaques formed by the enzymatic breakdown of amyloid precursor protein (APP) and neurofibrillary tangles that is occurred by hyper-phosphorylation and oligomerisation of tau in this region. Consequently, it leads to disruption in neuronal transmission due to the slowdown of enzymatic signalling and nutrient supply to the neurons resulting in a decrease in the count of neurons.78,79 In this study, the neuronal count found in CA1 and CA3 regions (out of 100) is given in Table 4. A considerable increase in the neuronal numbers was observed in groups IV, V, and VI compared to group II. However, neuronal numbers indicate a substantial protective role of formulated CLAA compared to CAE and CLCAE.\n\nAChE enzyme is responsible for the degradation of acetylcholine levels at a synaptic cleft region and influences the cholinergic neurotransmission. A single molecule of AchE can break down 5000 Ach molecules per second.80 In Figure 11, (n=6) normal control (group I) shows a significant increase in the AChE level in the brain, and the level of acetylcholine found decreased as compared to group I (p< 0.05). The accumulation of AChE in the normal control group forms a network with Aβ peptide and stimulates amyloid fibril formation in the hippocampus region.81 However, the positive control group showed substantial enhancement in acetylcholine levels by altering the active sites of AChE and inhibiting its activity. Group IV, V, and VI exhibited a significantly decreased AChE activity (p<0.05) and were found to have increased acetylcholine levels compared to group II. Furthermore, the group VI CLAA treated showed enhanced acetylcholine levels (p<0.05) compared to CAE treated group IV due to better bioavailability in the serum. The experimental facts herein revealed, a significant level of acetylcholine observed indicating that CLAA alters the enzymatic reaction of AChE and prevents the breakdown of Ach, which leads to the role of neuroprotection.82\n\nNote (bottom): Data represented as mean ± SD. Statistical analysis was performed one way ANOVA followed by Bonferroni multiple comparison tests. ##p< 0.001, * p< 0.01, b p < 0.1, c< 0.05 when compared to disease control (n=6).\n\nThe amount of drug that reaches into systemic circulations and the amount of drug absorption was determined. Herein, the oral bioavailability of optimised CLAA was compared with optimised CLCAE and AA (Table 5). The in vivo oral bioavailability of CLAA in the initial hours was less in the serum due to the packed chitosan coat and later exhibited better bioavailability in the serum that lasted up to 8 hours. AA showed a maximum concentration in the first four hours. The maximum serum concentration of AA was found to be 3.43±0.12 μg/ml at 4 hours, and the optimised CLCAE was found to be 5.32± 0.34 at 6 hours. The optimised CLAA achieved 9.23±0.34 μg/ml of serum concentration at 6 hours and was sustained for an extended period of time.83 It extended gut residence time by adhering to the intestinal mucosal layer by electrostatic interaction i.e., forming of a disulphide bridge between positively charged polymers with negatively charged cysteine-rich subdomains of mucus glycoproteins. It was found compared to the free drug that mucoadhesion enhances the portion of liposomal drug to passive permeation across the apical pole surface of intestinal epithelial.84 The formulation CLAA showed substantial improvement (p< 0.05) in oral bioavailability with pure AA and CLCAE.\n\nAs shown in Table 5, the pharmacokinetic parameters were estimated using the computer software PK/PD (Figure 12) (n =6). Tmax, Cmax, and elimination half-life values were greater in CLCAE and CLAA treated group than in serum from the AA-treated group. The optimised CLAA-treated rat serum showed lower values for the elimination rate constant, clearance, and volume of distribution. The optimised CLAA with a greater relative bioavailability of 75.56±0.6%, persisted for a longer duration in the body and the optimised CLCAE showed 53.23±0.3% relative bioavailability. A considerable improvement in the relative bioavailability of the CLAA took place as a result of the chitosan's bioadhesion activity, resulting in significant progress in the absorption of AA into the intestinal mucosa. In terms of time, there was a significant correlation between the rate of in vitro drug release and the in vivo plasma concentration of a drug. The formulations showed sustained drug release and a notable increase in drug bioavailability in the serum, possibly due to enhanced solubility and permeability compared to AA. The oral bioavailability of the optimised CLAA was significantly enhanced compared to optimised CLCAE and AA, which may be due to the decreased intestine and hepatic metabolism of the CLAA.85,86 An experimental study showed that optimised CLAA carries enormous potential to enhance the efficacy of phytoconstituents with greater accuracy. However, more research into the clinical trial and in vitro-in vivo correlation (IVIVC) might help it get to market faster.\n\nThe formulation stability data at 5±2°C, 32±2°C/60% ± 2% RH is specified in Table 6. A stability study could not be carried out at higher temperatures (> room temperature) because phospholipids in liposomes would deteriorate at higher temperatures.87 The results showed that after the third month, formulations kept at refrigeration temperature had better entrapment efficiency than all samples maintained at room temperature. The EE of optimised CLCAE varied with storage time and temperature. A considerable change in entrapment efficiency was observed and was found to be in the range of 51.3% to 49.5%, which indicated that liposomes were slightly stable. In contrast, throughout the stability study, the optimised CLAA exhibited a high degree of stability in entrapment efficiency. i.e., 71.2% to 71.2%. There was no difference in vesicle size in the optimised CLAA even after three months of storage (209.8 nm to 209.8 nm). The ζ potential of the optimised formulations exhibited minimal changes (+ 20.8 to + 22.2 mV), indicating excellent stability of liposomes. In the optimised CLCAE, a substantial difference was found in vesicle size compared to the CLAA. The vesicle size was slightly bigger after the second month of storage, which indicated that congeal liposomes subsequently affected the entrapment efficiency of the CLCAE.\n\n\nConclusion\n\nThe chitosan-coated liposomes of AA were found to be stable in the gastrointestinal tract GIT and controlled site-specific absorption. The developed novel product demonstrated an increase in AA's oral bioavailability compared to the conventional oral formulation as it showed great potential of extract when administered in modified form (CLCAE). CLAA also proved to be a better formulation than CLCAE because of AA’s uniform molecular size and potent pharmacological action. The CLCAE showed better pharmacological action than the standard extract of CA, attributed to the chitosan coating. Hence, the developed product can enhance efficacy and improve patient compliance through oral delivery. Furthermore, the modified, developed formulation can be commercialised as a nutraceutical product to prevent AD. The present work outcome is promising, and more experimental data and clinical study are required for further authentication of asiatic acid efficacy in nano-vesicular form for preventing or treating early stages of AD.",
"appendix": "Data availability\n\nFigshare: Data file 01 - In vitro evaluation data, https://doi.org/10.6084/m9.figshare.21484905.v1. 88\n\nFigshare: Data file 02 - In vivo data, https://doi.org/10.6084/m9.figshare.21485256.v1. 89\n\nData are available under the terms of the Creative Commons Attribution 4.0 CC-BY (Attribution)\n\nRepository: The ARRIVE guidelines 2.0 checklist and flow chart for “Cationic biopolymer decorated Asiatic Acid and Centella asiatica extract incorporated liposomes for treating early stage Alzheimer’s disease: An In-vitro and In-vivo Evaluation”. https://doi.org/10.6084/m9.figshare.21621282.\n\nData are available under the terms of the Creative Commons Attribution 4.0\n\n\nAcknowledgments\n\nThe NGSM Institute of Pharmaceutical Sciences in Mangalore and the Manipal College of Pharmaceutical Sciences at Manipal Academy of Higher Education (MAHE), Manipal, have generously provided the necessary resources for the conduct of this research, which the authors gladly recognize.\n\n\nReferences\n\nChen CS, Ouyang P, Yeh YC, et al.: Apolipoprotein E polymorphism and behavioral and psychological symptoms of dementia in patients with Alzheimer disease. 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}
|
[
{
"id": "159535",
"date": "11 Jan 2023",
"name": "Harishkumar Madhyastha",
"expertise": [
"Reviewer Expertise Nano medicine and Nutraceuticals"
],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address 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 \"Cationic biopolymer decorated Asiatic Acid and Centella asiatica extract incorporated liposomes for treating early-stage Alzheimer’s disease: An In-vitro and In-vivo investigation\" by Dubey and others describes the conjugated polymers with nutraceuticals for combating early stages of AD. Investigation hypothesis is well-designed and all supporting evidences for preparations, characterizations and drug loading validation and finally functional evaluation of conjugates are being studied. Before final acceptance for indexing I recommend the authors to attend to the following points in the revision:\nA hypothetical graphical abstract is required so that readers can quickly understand the whole theme in a single glance.\n\nAdditional information on the rationale of using centella asiatica extracts is required in the introduction; especially on phyto-mapping constituents of this plan (which is required).\n\nDid the investigators use whole plant including roots or some selective parts for extraction procedure?\n\nThe detailed procedures on formulation of conventional as well as chitosan-coated liposomes of CAE are required.\n\nReplace figure 2 with peak only representations.\n\nScale bar is missing in TEM (Fig. 4) of CLCAE and CLAA.\n\nFig. 10: what is the magnification?\n\nSome more points on the mechanism of actions of phytoconstitutents and AD is required in the discussion.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\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": "9219",
"date": "03 Mar 2023",
"name": "Srinivas Hebbar",
"role": "Author Response",
"response": "1. A hypothetical graphical abstract is required so that readers can quickly understand the whole theme in a single glance. Graphical abstract is submitted already while submitting the manuscript. Please see the PDF file linked here. 2. Additional information on the rationale of using Centella asiatica extracts is required in the introduction; especially on phyto-mapping constituents of this plan (which is required). Role of CA in AD. CA (Centella asiatica) is the most studied herb for its nootropic action. It is believed to be a promising agent for treating AD in the future because of its anti-amyloid property, cholinergic and antioxidant property. Oxidative damage is the main reason for many age-related disorders. Free radicals like hydroxyl (OH), hydrogen peroxide, peroxynitrite, and superoxide dismutase are responsible for cell damages during AD condition. CA extract (CAE) selectively decreases amyloid levels in the hippocampus of AD animal model. Dhanasekaran M et al (2009) have shown that CAE can impact the amyloid cascade altering amyloid β pathology in the brains of PSAPP mice and modulating oxidative stress components response that has been implicated in the neurodegenerative changes that occur with AD. The study resulted in a decrease in amyloid-beta with 2.5mg/kg of CA extract for the treatment period of 8 months. It also confirmed that CA extract acts as an antioxidant agent in-vitro by scavenging the free radicals and offers protection against DNA damage. Role of CA against D-galactose induced cognitive impairment, oxidative and mitochondrial dysfunction in mice were conducted by Anil Kumar et al. Chronic administration of D-galactose (100 mg/kg s.c.) for a period of six weeks significantly impaired cognitive task and oxidative defense (Increased lipid peroxidation, nitrite concentration and decreased activity of superoxide dismutase, catalase and non-protein thiols) and impaired mitochondrial complex (I, II and III) enzymes activities. The study concluded that six weeks CA (150 and 300 mg/kg, p.o) treatment significantly improved behavioural alterations, oxidative damage and mitochondrial enzyme complex activities as compared to control l (D-galactose). The role of CA fresh leaf extract treatment on the dendritic morphology of hippocampal CA3 neurons was studied by Gadahad et al . Rats (2.5 months old) were fed with 2, 4 and 6 ml/kg body weight of fresh leaf juice extract of CA for 2, 4 and 6 weeks, respectively. The results showed a significant increase in the dendritic length (intersections) and dendritic branching points along the length of both apical and basal dendrites in rats treated with 6 ml/kg body weight/day of CA for 6 weeks. However, the rats treated with 2 and 4 ml/kg body weight/day for 2 and 4 weeks did not show any significant change in hippocampal CA3 neuronal dendritic arborisation 3. Did the investigators use whole plant including roots or some selective parts for extraction procedure? For the extraction process, only leaf and stem part of the Centella asiatica plant was used. It was dried and extracted through Soxhlet apparuts. 4. The detailed procedures on formulation of conventional as well as chitosan-coated liposomes of CAE are required. Formulation of Chitosan coated liposome of CAE (CLCAE) The liposome of CAE (LCAE) was formulated by the solvent evaporation method, with some modifications. Different ratios of CAE, soya phosphatidylcholine (SPC) and cholesterol were dissolved in dichloromethane (20 ml) in 100 ml round bottom flask. It was subjected to the rotary flash evaporator (Superfit rotavap series, 6-BU, continental Pvt. Ltd., Mumbai, India), and the flask was rotated with 60 rpm speed at 55 °C to obtain a thin film. The film was hydrated with phosphate buffer pH 6.5 at room temperature. CLCAE was prepared as per the improved ionotropic gelation method. Chitosan was dissolved in acetic acid (0.5% w/v) and kept overnight at room temperature. An appropriate amount of chitosan solution was added drop-wise using a syringe to the optimised CAE liposomal suspension under continuous magnetic stirring at room temperature for one hour. It was undisturbed for three-four hour to get a proper swelling of the liposomes. 5. Replace figure 2 with peak only representations. Replaced (Not able to paste in this section). 6. Scale bar is missing in TEM (Fig. 4) of CLCAE and CLAA. The scale bar of the Fig 4. Is 200 nm. 7. Fig. 10: what is the magnification? The magnification of fig 10 is 40x 8. Some more points on the mechanism of actions of phytoconstitutents and AD is required in the discussion. Asiatic acid (AA) is a natural aglycone of pentacyclic triterpenoids of the ursane type. It is abundantly present in CA and many edible and medicinal plants. It is a reputed herb for neuropsychiatric disorders and wound healing in many traditional medicinal formulations. AA can modulate various enzymes, receptors, growth factors, cell signalling etc Role of AA in AD AD is a neurodegenerative disease caused due to multifactorial pathogenesis reactions, mainly oxidative stress and inflammation in the neuron triggers amyloid plaque formation. As per the study conducted by Jew et al (2000), AA was showed most effective 97% potent against amyloid plaque. AA modulates the enzymatical pathways such as BACE1, (Beta site APP cleaving enzyme 1) ADAM10,(A disintegrin and metalloproteinase 10),IDE (Insulin degrading enzyme), NEP (Neprilysin) BACE1 enzyme is responsible for regulating the formation of Aβ from Aβ precursor protein. However, ADAM10 controls the rate of non-amyloidgenic formation of AβPP. Effective degradation of Aβ was done using IDE and NEP enzymes. The AA inhibits the Aβ plaque formation by reducing BACE1 and regulates the enzymatic action of IDE, NEP and ADAM10 and also helps in neurogenesis. Aluminium ion is a neurotoxic molecule responsible for regulating AβPP AA shows neuroprotection against aluminium maltolate –induced neurotoxicity by inhibiting acetylcholinesterase, gamma secretases sideways with amyloid β 1-42 (insoluble protein) and glial fibrillary acidic protein. AA shows potent antioxidant activity inhibiting H2O2 induced cytotoxicity and reduces apoptosis by reducing BAX and capase 3 gene expressions responsible for ROS production (Reactive oxygen species)"
}
]
},
{
"id": "159536",
"date": "25 Jan 2023",
"name": "Manodeep Chakraborty",
"expertise": [
"Reviewer Expertise Molecular pharmacology"
],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThere is a need for more phytoconstituents-based studies in the pharmaceutical field. The assessments of the research gaps are also self-evident, and this study's methodology and tools have a higher relevance to concluding the successful formation of the surface-modified liposome.\nResults of the pre-formulation study on AA comply with the standard reported literature limits. The developed RP HPLC method was found to be straightforward, quick, precise, reliable, and consistent for determining the AA (13 min). The performance qualities did not significantly alter during validation.\n\nThe conventional liposome of AA was developed using a solvent evaporation method. It was a well-accepted method for the preparation of liposomes. Chitosan was reported to be useful in coating negatively-charged liposomal surface because of electrostatic interactions between the negatively charged SPC and the positive charges of primary amino groups of chitosan.The ionotropic gelation method was adopted for coating the conventional liposomes.\n\nFrom the statistical optimization using Design Expert® software (32 full factorial designs) the % error between predicted and actual was found to be less than 5 %; the report was found to be reliable.\n\nIn the H1 NMR investigation, the change in proton signals in the aromatic region clearly demonstrated the establishment of molecular bonds with AA. The phenolic region of AA and the combination of phospholipids have weak intermolecular contact. The average vesicle size of sonicated optimized CLCAE and CLAA were found to be 224.4 nm, 209.8 nm that signifies the optimum complex formation in which drug molecule tangibly bonded with the polar heads of lipid resulting in a reduction of surface negative charge and the vesicle size. The positive ζ charge of optimized CLAA shows good physical stability that ultimately favors the mucoadhesion property to the cell membrane. Studies using DSC and FTIR on CLAA that had been improved gave the first official evidence of the complexation between AA and SPC. TEM and AFM confirmed no evidence of aggregation or decomposition. But the scale was missing in the image. The optimized formulation had increased and sustained the drug release be due to physicochemical change and complexation between AA with SPC and chitosan.\n\nAlzheimer’s disease was successfully induced to experimental Wistar rats with the Alcl3 induced method which is a well-known method. The behavioral assessment was performed using the Y maze to confirm the disease progression. The scale in the histopathology image was missing. Both the histology examination and the neuronal count showed that the protection against disease progression was present.\n\nThe results of the experiment showed that the group treated with the optimized formulation had a considerable amount of acetylcholine. This suggests that CLAA functions as an inhibitor of the cholinesterase enzyme.\n\nThe results of the study are also openly admitted. The explanation related to the work was clearly written within its limitations and provides further direction and scope for research.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? 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": "159533",
"date": "30 Jan 2023",
"name": "Udit J. Chaube",
"expertise": [
"Reviewer Expertise Cancer",
"Cancer biology",
"Design and Synthesis of Novel Leads as mTOR inhibitors and CDK2 inhibitors"
],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis work aims to create and compare chitosan-coated liposomes of Asiatic acid (CLAA) and Centella asiatica extract (CLCAE) for increment in oral absorption and bioavailability in the treatment of ADs in early phases.\nThe method of solvent evaporation was used by the authors for the development of CLAA and CLCAE.\nThe ALCl3-induced Alzheimer's Disease Model was used to further assess this formulation, and the results showed that CLAA has better stability and was the optimal oral drug delivery mechanism.\nAlthough, there are certain gaps in the pharmacological evaluations which can be rectified. For Example: Missing of Scale on the Histopathological Slide. In the future, if the authors are carrying out this investigation, I urge them to employ Amyloid beta (A) rather than AlCl3 for the induction of AD. Additionally, the authors should use western blot analysis and gene expression studies in their upcoming research project (Explore Molecular Targets of Alzheimer's Disease), which would ultimately add innovation to the 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? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes",
"responses": []
}
] | 1
|
https://f1000research.com/articles/11-1535
|
https://f1000research.com/articles/11-1153/v1
|
10 Oct 22
|
{
"type": "Research Article",
"title": "Mathematical modeling of HIV-HCV co-infection model: Impact of parameters on reproduction number",
"authors": [
"Oluwakemi E. Abiodun",
"Olukayode Adebimpe",
"James A. Ndako",
"Olajumoke Oludoun",
"Benedicta Aladeitan",
"Michael Adeniyi",
"Olukayode Adebimpe",
"James A. Ndako",
"Olajumoke Oludoun",
"Benedicta Aladeitan",
"Michael Adeniyi"
],
"abstract": "Background: Hepatitis C Virus (HCV) and Human Immunodeficiency Virus (HIV) are both as classified blood-borne viruses since they are transmitted through contact with contaminated blood. Approximately 1.3 million of the 2.75 million global HIV/HCV carriers inject drugs (PWID). HIV co-infection has a harmful effect on the progression of HCV, resulting in greater rates of HCV persistence after acute infection, higher viral levels, and accelerated progression of liver fibrosis and end-stage liver disease. In this study, we developed and investigated a mathematical model for the dynamical behavior of HIV/AIDS and HCV co-infection, which includes therapy for both diseases, vertical transmission in HIV cases, unawareness and awareness of HIV infection, inefficient HIV treatment follow-up, and efficient condom use. Methods: Positivity and boundedness of the model under investigation were established using well-known theorems. The equilibria were demonstrated by bringing all differential equations to zero. The associative reproduction numbers for mono-infected and dual-infected models were calculated using the next-generation matrix approach. The local and global stabilities of the models were validated using the linearization and comparison theorem and the negative criterion techniques of bendixson and dulac, respectively. Results: The growing prevalence of HIV treatment dropout in each compartment of the HIV model led to a reduction in HIV on treatment compartments while other compartments exhibited an increase in populations. In dually infected patients, treating HCV first reduces co-infection reproduction number Rech, which reduces liver cancer risk. Conclusions: From the model's results, we infer various steps that policymakers could take to reduce the number of mono-infected and co-infected individuals.",
"keywords": [
"Mathematical model",
"HIV/AIDS",
"HCV",
"infection-free equilibrium",
"unawareness",
"awareness",
"endemic equilibrium",
"next generation matrix",
"basic reproduction number",
"stability."
],
"content": "Introduction\n\nEmerging and reemerging infectious illnesses are of public health importance, and mathematics has traditionally been employed to acquire a realistic understanding into the transmission dynamics and control of these diseases. Both Hepatitis C Virus (HCV) and Human Immunodeficiency Virus (HIV) are considered blood-borne viruses because they are spread through contact with the blood of an infected individual.1 In 2017, 2.3 million people living with HIV were simultaneously infected with HCV, according to the World Health Organization (WHO, 2016). Infectious diseases like HIV and HCV have become critical problems in public health around the world. Africa and South and East Asia bear the heaviest brunt of these co-infections (WHO, 2017). Co-infection with HIV and another disease usually poses greater dangers and has more dire outcomes for individuals. When HIV is present alongside HCV, the viral infection advances much more quickly in the latter. If the CD4 cells is less than 200 cells/mm3, the risk of severe liver injury increases.2 Hepatocellular carcinoma, liver cirrhosis, and liver-related mortality are also more likely to occur.3 The international community agrees that strong leadership in the form of well-thought-out programs and policies that focus on prevention, early diagnosis, therapies that respects patients' rights, and high-quality, universally accessible health care is needed to stop the spread of HIV. Concerning co-infection, there have been reports of effective HCV drug combinations in treating people who are both HIV positive and HCV positive. Furthermore, HIV can be treated successfully in the majority of people with HCV.4 New antiviral medications have the potential to treat HCV in persons who are HIV-positive and infected with HIV, but additional research is needed to prove their effectiveness.\n\nThere are about 40 million PLHIV in the world right now. UNAIDS, the United Nations Program on HIV/AIDS, estimates that in 2020, more than one person every minute would die from an AIDS-related illness.5 HIV and HCV can be spread in many ways, such as through injections, sexual contact, and being passed down from parent to child.6 People with HIV often also have HBV and/or HCV.7,8 One of the main reasons people with HIV die is because of liver disease.9,10 There are over 2 million PLHIV on a global scale who are living with HBV or HCV.1,7,8 Bi-directional effects explain why people who have HIV who also have HBV and/or HCV have a greater risk of becoming sick and die. HIV patients with HBV and/or HCV quickly develop AIDS,11 and antiretroviral drugs are more harmful to the.12–14 On the other hand, when PLHIV change their immune response, it leads to less HCV viral clearance, reactivation, and replication in co-infected individual. Aspartate aminotransferase, alanine aminotransferase, and alkaline phosphatase levels rise as a result, and chronic liver disease complications like cirrhosis, hepatic decompensation, and hepatocellular carcinoma as well as a higher death rate15–17 progress more quickly. People living with HIV who also have HBV, HCV, or both have a greater risk of infection transmission. However, there has been relatively little deterministic study of HCV chronic infection co-infected with HIV. For instance, Ref. 18, introduced and analyzed a deterministic model for HCV and HIV co-infection. Focusing on HCV and HIV co-infection, they hope to better understand the long- and short-term dynamics of both diseases and develop methods for forecasting whether HCV and HIV will eventually become extinct or remain a persistent problem. To ascertain the effect of treatment on the dynamics of each disease, in19 built and investigated a mathematical model of the co-dynamics of the HCV and HIV/AIDS. The equilibria (disease-free and endemic) are described under which they are both locally and globally asymptotically stable. Similarly, in Ref. 20, investigated mathematical model of co-infection with HIV and HCV. In the case of HIV, the innovation of their strategy is the incorporation of therapy for both infections as well as how it is passed from mother to kid.21 Constructed a mathematical model of HCV/HIV co-infection within the host by modifying a model of HCV mono-infection that had previously been published to include an immune system component in infection clearance. They then combined a decline in immunological function with an increase in HIV viral load to examine the impact of HIV co-infection on spontaneous HCV clearance and sustained virologic response (SVR). Also, Ref. 22, through mathematical, created a new co-infection model for the hepatitis C virus (HCV) and human immunodeficiency virus (HIV) (HIV). Examining therapy for both diseases, Additionally, using mathematics,22 developed a new co-infection model for the human immunodeficiency virus (HIV) and hepatitis C virus (HCV) (HIV). Examining prevention, diagnosis, screening, HIV knowledge and awareness, condom use, and largely using numerical simulations, ignorance and awareness, and condom use and mostly employs numerical simulations. In, Ref. 23, constructed two ODE models at the population level to mimic the progression of the HCV and HIV among PWID. Both deterministic and stochastic solutions were used to solve the models describing HCV and HIV parenteral transmission. Additionally, several deterministic models that are relevant to our work have been suggested and examined in Refs. 24–29.\n\n\nThe HIV and HCV co-infection model\n\nThe paradigm of co-infection between HIV and HCV is described in this section.\n\nWe determine overall and submodel reproduction rates (HIV only and HCV only models). We investigate global and full model disease-free equilibrium local stability. We determine the reproduction number's sensitivity indices to important model parameters. Simulation diagrams created using Runge-kutta order four embedded in maple 2020.1 software and contour plots created using maple 2020.1, help understand the model's dynamics.\n\nThe mathematical model that will be considered and investigated is divided into (15) different groups, namely, the susceptible populace for both HIV and HCV St, the HIV-infected unaware Hut, the HIV-infected aware, HAt, HIV on treatment HTt, the AIDS populace aware and on treatment AAt, acutely infected Ict and chronically infected Cct infected HCV, HIV-unaware co-infected with acute and chronic HCV (HuItandHuCt), HIV-aware co-infected with acute and chronic HCV (HAItandHACt), HIV-positive individuals receiving treatment for HIV who are co-infected with acute and chronic HCV (HTItandHTCt), HIV-positive individuals in stage-IV co-infected with acute and chronic HCV AAItandAACt.\n\nThe overall population at time t, represented by Nt,is classified into the 15 classes/subgroups listed in Tables of Nomenclature, each of which corresponds to a different epidemiological status.\n\nIn Figure 1, the epidemiology of co-infection with HIV and HCV is depicted schematically. The many compartments (circles) symbolize the various disease phases, and the arrows depict how people progress from one phase to the next. At time t, susceptible individuals S are assumed to enter the population at a constant rate, 1−φHuΛ. Some newborns acquire HIV at parturition and are subsequently enrolled directly into the infectious class, Hu where φ, is the rate of newborn HIV infection and Λ is the rate of recruitment through immigration or emigration. Individuals in all classes die at a consistent natural mortality rate, μ. Individuals with AIDS AaAaIAaC have an extra death rate owing to AIDS, ⅆa.We assume that HIV-infected people who are receiving treatment do not spread the virus.30,31 Despite the complexity of disease co-dynamics, we will make the simple assumption that co-infected and mono-infected people can only transmit one of the two diseases—HIV or HCV—at a time. Individual S, who is susceptible to HIV infection, is at risk of acquiring HIV infection at a rate of λH, (force of infection related to HIV) when in contact with the HU,HA,andAa populations, where\n\nThe parameter bh is the chance that a person will get HIV from a contact, and the parameter, the average annual number of sexual partners for someone at risk of contracting HIV is ch. To highlight the usage of condoms as a crucial prevention measure, We presume that ψξ∈01indicates the degree of condom protection. If ξ=0, condom use offers no protection, ξ=1 denotes perfect protection, where ψ is the use of a condom.\n\nWhen compared to persons who are only infected with HIV, the relative infectiousness of persons who are acutely infected with HCV and unaware of their HIV infection HUI and individuals who are chronically infected with HCV and AIDS AAc, is accounted for by the parameters κ1>1. We make the assumption that persons who are co-infected are approximately three times more infectious than individuals who just have one infection.32,33 HIV unaware class ,HU,HUI,HUC singly and dually infected with HCV advances to HIV diagnosed class HA,HAI,HAC after testing at a rate, α1,α2,α3and those in aware HIV was enrolled on therapy at the rate θ1,θ3,θ5 in class HT,HTI,HTC.Nevertheless, some individuals who were placed on HIV treatment default from or drop out of the HAART treatment25 after which they develop AIDS due to drug resistance and progress to class AA,AAI,AAC at a rate υ1,υ2,υ3. People with HIV and HCV who don't know their HIV status, HU,HUI,HUC and didn't get tested move to the AIDS class AA,AAI,AAC.at a rate ρ1,ρ2,ρ3, People with AIDS symptoms singly and dually infected with HCV are given treatment at a rate of θ2,θ4,θ6 respectively. AIDS infected can respond well to treatment and return to HT,HTI,HTC18,20 and die because of AIDS at an incidence da.\n\nSusceptible people get HCV infection from people in the Ic,Cc,HUI,HUCat a rate of λC where λCis the risk of getting HCV, which is given by\n\nTo simulate the reality that individuals who are dually infected are more infectious than the mono-infected, we use the notation κ2>1, where bcthe likelihood that contact will result in HCV infection.19,34,35\n\nPeople who are only infected with HIV HUHAHTandAa acquired HCV at a rate δ1λcδ2λcδ3λc and moved to classes HUIHAIHTIAAI, an increased risk of HCV acquisition is accounted for by the modification parameter δ1,δ2,δ3>1. HCV-only infected people IcCcare more likely to obtain HIV HuIHuC than people who are only infected with HCV at a rate γλH,τλH where γ,τ>1translates to an increased chance of contracting HIV for people whose immune systems are weakened by HCV.\n\nHIV and AIDS patients, dually infected with the acute HCV HUI,HTI,AAI;at a rate η, becomes chronically infected and are treated for chronic HCV epidemic at rii=12… while the remaining populace ,ω spontaneously clear the virus to return to susceptible class S. We then assume that an individual whose immune system helps in clearing the virus can become re-infected at rate λC if expose or engage in risk behaviors such as injection drug use,33 drinking alcohol,36 multiple sex partners and sex between two men37 since the clearance does not confer permanent immunity.38\n\nAn HCV-positive person stays acutely infected for an average of 1/σc days. Since newer combinations of direct-acting antivirals (DAAs) have showed high cure rates of 90%-95% in phase II and III clinical trials, we did not take treatment failure for chronic HCV carriers into account. However, researchers are beginning to report sporadic incidences of treatment failure in HCV.33,39,40\n\nIn people with HIV and HCV co-infection, little is known regarding the relationship between spontaneous HCV clearance and sustained HIV infection control.41 Co-infection reduces the possibility of the acute HCV virus clearing itself naturally.33,42,43 Because HIV speeds up the development of HCV, a high viral load for this virus may also indicate a rapid progression of liver disease.33,37,43 In order to take into account the additional viral load resulting from co-infection, we use the term ε1 to impact spontaneous clearance and the term ε2to accelerate the disease progression, due to co-infection.30 Due to the fact that HCV and HIV-1 are spread through the same ways, about 10–15 percent of acute HCV infections clear up on their own, but less than 10 percent of HIV-1 infections do. The compartmental flow diagram for the HIV-HCV co-infection model is depicted in (Figure 1).\n\nMathematically, the flow chart leads to the 15 systems of ordinary differential equations listed below:\n\n\n\n• People who are being treated for HIV don't spread the virus.\n\n• Co-infected people are approximately three times more contagious than mono-infected people.32\n\n• Persons co-infected with HIV who were not getting ART were presumed to spread HCV more easily due to higher viral loads.\n\n• Proportional (random) mixing between all groups.\n\n• It is assumed that an individual could be re-infected with HCV even after successful treatment if expose or engage in high-risk behaviors such as injecting drugs,33 drinking alcohol,36 having multiple sex partners and sex between two men37 since the clearance & treatment does not confer permanent immunity.39\n\n• Treatment failure for people who have had HCV for a long time isn't taken into account because recent research has shown that newer combinations of direct-acting antivirals (DAAs) have shown cure rates of 90% to 95% in phase II and III clinical trials.33\n\n• Individuals acutely infected with HCV were assumed to spontaneously clear the virus.44\n\n• Mono-infected and co-infected people can transmit either HIV or HCV, but not both simultaneously.\n\nSince Equation (4) represents a population of humans, all of the corresponding parameters are positive. The subsequent non-negativity finding is also valid.\n\nHIV and HCV will be analyzed independently. Thereafter, the co-infection analyses will be carried out, with positive initial conditions specified by;\n\nAs a result, the system dynamics (3.4) will be examined in light of the biological elements of the region\n\nThe system variables (1) are positive whenever t > 0. In other words, Solutions of the system (4) with a positive initial condition will remain positive for every t > 0.\n\nLet Φ=sup{St≥0,Hut≥0,HAt≥0,HTt≥0,Aat≥0,Ict≥0,Cct≥0,HuIt≥0,HAIt≥0,HTIt≥0,AAIt≥0,HUCt≥0,HACt≥0,HTCt≥0,AaCt≥0.The regionΦ∈ℝ+15\n\nIt follows from the model's first equation (1) that\n\nwhich is re-writeable as\n\nHence,\n\nSo that\n\nThus, S≥0\n\nAnalogously, it's easy to show that\n\nfor all t>0,are all positive.\n\nThe closed set Φ={St+Hut+HAt+HTt+Aat+Ict+Cct+HuIt+HAIt+HTIt+HUCt+HACt+HTCt+AAIt+AaCt∈ℝ+15:N≤Λμ} is positively invariant.\n\nNow we demonstrate that every possible solution is uniformly bounded in. By adding all system (4) equations, we obtain:\n\nIt follows from the equation that limt→∞supNt≤Λμ. As a result, the system's dynamics (4) will be looked at in light of the region’s biological factors. This is simple to demonstrate as being positively model-invariant.\n\nTherefore as t⟶∞, Λμ is the upper limit of N given that N0≤Λμ, Ntwill decline to this level if N0>Λμ. As a result, the region Φ contains all possible system solutions that can enter or remain. Under the flow caused by the system (4), the region of biological interest Φ is therefore positively invariant Therefore, since region Φ is positively invariant and the results for the system's existence and uniqueness hold there, it is sufficient to analyze the dynamics of the flow caused by the model (4) in region Φ.\n\n\nPoints of equilibrium, reproduction numbers and the stability analyses\n\nIn this section, computation of disease-free equilibrium (DFE) and the endemic equilibrium (EE) will be carried out, and their stability will be examined using associative reproduction number.\n\n\nDisease-free equilibrium and the effective reproduction number\n\nIn this part, we calculate model R0′s RN.\n\nThe effective reproduction number ReHC, is known as the spectral radius of the next generation matrix,46 governs EoHC′s linear stability. In the presence of a strategic intervention, the effective reproduction number is frequently understood as the estimated number of secondary infections produced by a single infectious individual during his/her entire infectious phase. Nevertheless, in the suggested model, the infectious persons can be classified into any of these fourteen classes HU,HA,HT,AA,IC,CC,HUI,HUC, HAI,HAC,HTI,HTC, AAI,AACwith the estimated count of secondary infections varying according to the class. Model's effective reproduction number (the total sum of secondary infections caused by HIV or HCV infected individual throughout the full contagious period in the context of treatment) is given using the latter technique as.\n\nWe will now estimate the reproduction number, ReHC, of the entire model (4). Model (4)'s infection-free equilibrium state E0HC is given by:\n\nOn system (4), we evaluate the matrices for the new transmittable terms F, the terms V, and matrix FV−1, based on submission in (1) – (4) above. The reproduction number is then the spectral radius of FV−1. R0HC is given after some mathematical manipulation (please see the Appendix for a complete proof):\n\nWhere k1=μ+α1+ρ1,k2=μ+θ1,k3=μ+ν1,k4=μ+da+θ2,k5=μ+dc+r1\n\nThe following lemma is derived from Theorem 2 of Ref. 46.\n\nIf R0HC<1, the disease-free equilibrium EoHC is asymptotically stable locally, otherwise it is unstable.\n\nBy evaluating the two model sub-models listed below.\n\nModel (9) is obtained from model (4) by equating to zero the variables pertaining to HIV dynamics HU=HA=HT=AA=HUI=HUC=HAI=HAC=HTI=HTC=AAI=AAC=0, while model (12) is developed from model (4) by setting to zero the variables pertaining to HCV dynamics (IC=CC=HUI=HUC=HAI=HAC=HTI=HTC=AAI=AAC=0). We now compute the system's reproduction number, RHIV (5). We employ the method of the next generation matrix in Ref. 46.\n\nWhere λH=c1−ψξbhHU+AANh, with total population given as\n\nDisease-free equilibrium (DFE) evaluation of F and V generational matrices is given by\n\nUsing Ref. 22, the new infection terms matrices F, and the terms, V, are as follows:\n\nThe matrix FV−1′s eigenvalues are as follows:\n\nThe associative basic reproduction number is stated as:\n\nwhere ρ stands for spectral radius of FV−1. The following lemma is derived from Theorem 2, Ref. 46.\n\nIf ReH<1, the disease-free equilibrium EoH is asymptotically stable locally, otherwise it is unstable.\n\nWe then derive the reproduction number, ReC, of model (12).\n\nWhere λC=c1−ψξbcIC+CCNc,where Nc is the total number of people given as\n\nA state of HCV-free equilibrium for the system of equations in (12) is obtained by:\n\nUsing Ref. 22, the new infection terms matrices F, and the terms, V, are thus:\n\nThe matrix FV−1′s eigenvalues are as follows:\n\nThe associative basic reproduction number is written as:\n\nIf ReC<1, disease-free equilibrium EoC is asymptotically stable locally, otherwise it is unstable.\n\n\nThe endemic equilibria and stability\n\nThe following endemic equilibrium states are available in model system (4):\n\nFrom model (4), we set to zero variables pertaining to HIV dynamics HU=HA=HT=AA=HUI=HUC=HAI=HAC=HTI=HTC=AAI=AAC=0, and is given by\n\nEC∗=S∗IC∗CC∗000000000000with\n\nWhere g1=ημσc+ηdcσc+μ2+μr+μdc+ηdcσcησc+ωσc+μr+μ+dc\n\nThe unique endemic equilibrium E c is said to be globally asymptotically stable for model system (4) if RC>1 and RHC<1.\n\nThere is no HIV in the community, so all of the HIV compartments have a value of 0. The Jacobian matrix of this three-dimensional system at endemic equilibrium S∗IC∗CC∗, is written as\n\nAs a result of traceJ being negative and the determinatJ being positive, the steady state is locally asymptotically stable. In order to demonstrate EC∗′s global stability, firstly we observe the domain SICCC≥0S+IC+CC<Λμ is positively invariant and attractive for the 3D system. Adopting Bendixson and Dulac's negative criterion to eliminate the presence of the periodic orbits using the expression 1ICand1CC as the Dulac multiplier, we obtain\n\nWhen the right side of the first equation is differentiated with regards to S, the second equation with regards to Icand the right side of the second equation is differentiated with regards to Cc,\n\nAs the sum of these three expressions are negative, periodic there is no existence of preriodic orbits. Consequently, Ec is globally asymptotic for Rc>1andRHC<1.\n\nThis occur by setting to zero the variables pertaining to HCV dynamics (IC=CC=HUI=HUC=HAI=HAC=HTI=HTC=AAI=AAC=0 and is given by Sh∗,HU∗,HA∗,HT∗,AA∗,0,0,0,0,0,0,0,0,0,0 which is present when R0>1 exists, the endemic steady states can be computed. so that,\n\nWe want to consider how the reproduction number of HCV, RC and reproduction number of RH impact one another as follows:\n\nwhich is the total sum of new HIV infections that one person with HIV will cause in a population where HCV is already common. Even if RC>1>RH, HIV will be allowed to spread into a population where HCV is common if RHC is greater than 1. In other words, RHC>1 which shows the presence of HCV makes it easier for HIV to spread in a community. But for RHC<1, HCV is still the biggest health issue, even though HIV has been spread to a population where HCV was already common and vice versa.\n\nTaking the partial derivative of RHC with regards to bh, we have\n\nAny time Rc>1, equation (14)'s positive result shows that the existences of HCV accelerates the spread of HIV infections in a community and vice versa.\n\nFrom (21) since the partial derivatives with respect to RC is positve, this signifies that as the reproduction number of HCV, RC increases, it impacts the reproduction number of HIV RH. Then, we should simply allow HCV infection to reduce to avoid increased viral load in HIV-infected individuals because any slight increase in HCV will make HIV increase.\n\n\nThe global stability of the disease free equilibria\n\nComputation of global stability of the disease-free equilibrium of the whole model (4) is done in this section. To start, we will calculate the stability of the disease-free equilibria of both of the sub-models (9) and (12).\n\nDisease-free equilibrium E0His globally asymptotically stable for model (9) if R0H is less than 1.\n\nHere, the Comparison theorem as outlined by Refs. 48–50 is applied. The rate of change of the system's infected components (9) can be expressed as:\n\nSince the disease-free HU=HA=HT=AA=0→0000 and Sh≤Nh, as t→∞ in Γh, F and V are defined as described for system (9) in section 2.2.1. Thus,\n\nIf R0H<1, then ρF−V<1, which is the same as stating that all eigenvalues of the matrix F−Vlie in the left-half plane.46 Therefore, the linear system described by the equality (23) is stable anytime R0H<1 and HU=HA=HT=AA=0→0000ast→∞for this linear ordinary differential equation (ODE) system. As a result of employing a basic comparison theorem,49–51 we obtain HU=HA=HT=AA=0→0000for the nonlinear system (9) represented by the last four equations of the system. We construct a linear system with St=Λμ by inserting HU=HA=HT=AA=0 into the first equation of model (9). Thus, StHUHAHTAA→Λμ0000ast→∞ for R0H<1, so E0His asymptotically stable globally if R0H<1.\n\nNow, we follow the same approach to compute the global stability of the disease-free equilibrium of the sub model (9).\n\nIf R0C<1, the disease-free equilibrium E0Cin submodel (9) is globally asymptotically stable.\n\nHere, the Comparison theorem as outline by Refs. 49, 50 is applied. The rate of change of the system's acute and chronic components (8) can be expressed as:\n\nIf R0C<1, then ρF−V<1, which is the same as stating that all eigenvalues of the matrix F−Vlie in the left-half plane. Therefore, the linear system described by the equality (12) is stable anytime R0C<1 and IC=CC=0→00ast→∞for this linear ordinary differential equation (ODE) system. As a result of employing a basic comparison theorem,49,51 we derived IC=CC=0→00for the nonlinear system (12) represented by the last two equations of the system. We construct a linear system with St=Λμ by inserting IC=CC=0into the first equation of model (12). Thus, StICCC→Λμ00ast→∞ for R0C<1, so E0Cis asymptotically stable globally if R0C<1.\n\nModel (4)'s disease-free equilibrium can only be globally stable under very narrow circumstances, namely when new co-infection cases are avoided. In such circumstances, patients with HIV or HCV infections could not get both diseases.\n\nThe global asymptotically stable HIV-HCV disease-free equilibrium E0 of the system (4) is unstable if RHC>1 and stable if RHC<1.\n\nThe Refs. 49, 50 Comparison approach is employed here.\n\nCheck appendix B for the proof of the GSA of the full model.\n\n\nNumerical simulation\n\nIn this part, we use the Maple computer language to perform in-depth numerical simulations to assess the effects of HCV treatment and antiretroviral therapy in dual-infected populations under various beginning conditions. Table 2 lists the parameter values we utilize for our numerical simulations.\n\nSelecting 100 different initial conditions, Figure 2 show that the trajectories of the solutions converge to (145, 0, 0, 0, 0), Hence, ReH=0.712, this aids the result in Lemma 4 that the disease-free equilibrium is globally asymptotically stable if ReH<1 in section 2.2.3. Also, the endemic equilibrium trajectories of the solutions converge to (8.420;22.353;17.485;91.452;4.534): in Figure 3 choosing different initial conditions, for a given parameter values and initial conditions given in Table 2 respectively, hence ReH=7.1234. This again supports Lemma 5 in section 2.2.3 that the endemic equilibrium is globally asymptotically stable if ReH>1:\n\nFigure 4, shows the behavioural dynamics of the HCV populations when Rec<1. Over time, a gradual increase in the susceptible population is obtained which later remains stable and does not tend to zero while acute HCV and chronic HCV tend to zero when Recis less than unity. This is an indication that the susceptible population will never be zero and endemicity will not exist. As such the disease will die over time due to the basic reproduction number of less than one, and the trajectories of the solution converge to 200,00,hence Rec=0.101 which authenticates the analysis shown in section 2.2.3 Lemma 5, that the disease-free equilibrium is globally asymptomatically stable if Rec<1. This indicates that disease dies out early which is influenced by effective condom use and other strategies.\n\nThe behavioural dynamics of the susceptible, acute HCV and chronic HCV populations in endemic states was shown in Figure 5. Each system approached asymptotically the stable HCV endemic equilibrium state of system 12. Moreover, the endemic equilibrium trajectories of the solution converge to 234.034,120.89489.469by choosing different initial conditions for given parameters in Table 2, hence, Rec=1.011. This again aids Theorem 3.12 that the endemic equilibrium is globally asymptomatically stable if Rec>1.\n\nFigure 6, shows the impact of fall-out on the HIV reproduction number, ReH. As the proportion of the fallout population increases HIV reproduction also increases. For example, if the proportion of the population that fall-out of treatment is 16.4%,ReH=0.04,ifυ=30%,ReH=0.042and whenυ=50%ReH=0.044, this supports the data fitting done by Ref. 47. Figure 7 shows the impact of fall-out on the dually infected with HIV-HCV reproduction number. As the proportion of the fallout population increases HIV reproduction also increases. For example, if the proportion of the population that fall-out of treatment is 16.4%,ReH=0.04,ifυ=30%,ReH=0.042and whenυ=50%ReH=0.044, this supports the data fitting done by Ref. 47.\n\nFigures 8-11 show the impact of vertical transmission on the dynamics of the HIV/AIDS infected classes. From these figures, even with a 2% increment in the population, there is a significant increase in the dynamics of the infected class. Figure 12 gives the impact of treating HCV first on the HIV-HCV co-infection population. The linear contour plot shows that when (0.60) 60% of the co-infected individual is treated for HCV the reproduction number Rechis0.6161%, also if we treat (0.80) 80% of the individual who are co-infected of their HCV first the Rechreduces to 0.55 (55%). The plot depicts that if we treat more of the dually infected population with HCV first, the transmission rate of the co-infection will be reduced by 0.14% thereby lowering the danger of liver cancer and death due to HIV/AIDS or death due to HCV. Likewise, Figure 13 depict the impact of treating HIV first on the HIV-HCV co-infection population. The linear contour plot shows that when (0.60) 60% of the co-infected individual is treated for HIV the reproduction number Rehcis0.90690.6%, also if we treat (0.8) 80% of the individual who are co-infected of their HIV first the Rehcreduces to 0.725 (72.5%). The plots depicts that treating more of the dually infected population with HCV first, the transmission rate of the co-infection more than treating HIV first in co-infected patient, which thereby lowering the danger of liver cancer and death due to HIV/AIDS or death due to HCV.\n\nFigure 14 described the impact of testing on the HIV-HCV co-infection population. The plot shows that when (0.30) 30% of the co-infected individual is tested for HIV the reproduction number Rehcis0.91691.6%, also if we test (0.6) 60% of the individual who are co-infected of their HIV first the Rehcreduces to 0.321 (32.1%). This shows that the more we test, the lower the risk of transmitting HIV and HCV.\n\nIn Figure 15, the effect of treatment and condom use on HCV reproduction numbers for the HCV model was shown on a contour plot. From the plot, if the treatment rate, r is 100% and the use of condoms is 90% it means that the reproduction number of HCV, Rec=0.0313. Likewise, if 57% of the population is treated and 77% of the population use condoms Rec will be Rec=0.0626compared to when 0.7% of the HCV infected population is treated while 10.4% used the condom then Rec rises to 0.250. This implies that to reduce the incidence of HCV transmission by the values of reproduction number, there is a need for more successful treatment where people attain SVR and avoid risk factors such as unprotected sex by use of condom, drinking, and multiple sexual partners which can make them re-infected. In Figure 16, the impact of the HCV reproduction number on the HIV reproduction number for system (4) is shown on a contour plot. From the figure, it is seen that when 20% of the population is infected with HCV, 9% of the population is been infected with HIV, then the reproduction number of the co-infection, Rech will be 0.0864 (8.64%). In the same manner, if we repeat 20% of the HCV population and 20% of HIV then we have Rech to be 0.201 (20.1%). This simply means that as the reproduction number for HCV, Rec increase it, in turn, increase the reproduction number of HIV Reh. Similarly, in Figure 17, the Impact of HIV reproduction number on HCV reproduction number is represented by a contour plot. Just as seen in Figure 16. When we have 10% of the HIV population, there are 8.1% of the HCV population and the co-infection Rech is 0.0861 (8.61%), Also, when 20% of the HIV are in the population and 2.73%of the HCV in the population, therefore we have Rech to be 0.201 (20.1%). This also means that as HIV increase in the population, HCV also increase. This simply implies that to control HCV, HIV cases will be reduced which is attributed to the same transmission process and it is vice versa. Hence to ensure the extinction of the co-infection in the population, if HCV is reduced it will in turn impact HIV and together if the two viruses RecandReh are low then there will be a reduction in the co-infection reproduction number, Rech.\n\n\nConclusion\n\nIn this study, we developed and studied a mathematical model for the dynamical behavior of both HIV/AIDS and HCV co-infection, which incorporates therapy for the two diseases, vertical transmission in HIV cases, awareness and unawareness of HIV infection, inefficient follow-up of HIV on treatment, and efficient condom use.\n\nThe stability analysis of the endemic equilibria revealed that: whenever the reproduction number is less than one, the unique disease-free equilibrium is both locally and globally asymptotically stable. Also, whenever the reproduction number is greater than one, the HCV-free endemic equilibrium is both globally and locally asymptotically stable. The examination of reproduction rates indicates that HCV treatment has a positive effect on HCV and HIV-HCV co-infection reduction.\n\nThe results suggest that policymakers should consider specific measures to minimize HIV infection, such as: developing campaigns to warn individuals about the consequences of having multiple sexual partners; distributing more condoms to individuals; continuing treatment for chronic HCV and AIDS and pursuing the inquiry of new and better drugs to combat HIV; treating infected newborns with HIV and advising pregnant women about the advantages of HIV counseling and testing, treatment; and treating newborns infected with HIV. Regarding HCV infection, therapy and other measures (e.g., greater promotional awareness about the disease and its transmission methods, among others) are highly suggested so as to achieve reduction in the number of chronic carriers and infectious.\n\nDespite the fact that this outcome is purely determined by the parameter values, it nevertheless implies that greater HCV transmission fuels HIV/AIDS and its development, hence playing a key part in the latter's increasing widespread. The same may be said for the influence of HIV/AIDS on HCV, as both HIV/HCV diseases exacerbate one another. Thus, treatment of HCV cases in areas with high HIV/AIDS prevalence will mitigate the impacts of HCV on HIV/AIDS epidemics and vice versa. Simulations indicate that the treatment of HCV has the potential to significantly minimize the detrimental result of HCV on HIV/AIDS epidemics.\n\nFuture research will investigate the impact of needle sharing on HIV and HCV transmission rates, as well as the application of the model to actual Portuguese data and calculation of its parameters.\n\nTherefore, it is possible to reduce the burden produced by HIV and HCV infection and their co-morbidity.\n\n\nData availability\n\nData used in this research can be found in Table 2: Parameters used in the numerical simulations of model.\n\n\nSoftware availability\n\nSource code available from: https://github.com/OE-Abiodun/HIV-HCV-COINFECTION-SIM-CODE/releases/tag/v3.0.0.\n\nArchived source code at time of publication: https://doi.org/10.5281/zenodo.6908227.57\n\nLicense: GPL-3.0 license",
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PubMed Abstract | Publisher Full Text\n\nAbu-Raddad LJ, Schiffer JT, Ashley R, et al.: HSV-2 serology can be predictive of HIV epidemic potential and hidden sexual risk behavior in the Middle East and North Africa. Epidemics 2010; 2: 173–182. PubMed Abstract | Publisher Full Text\n\nElbasha EH: Model for hepatitis c virus transmissions. Math. Biosci. Eng. 2013; 10(4): 1045–1065. PubMed Abstract | Publisher Full Text\n\nIngiliz P, Martin TC, Rodger A, et al.: HCV reinfection incidence and spontaneous clearance rates in HIV-positive men who have sex with men in Western Europe. J. Hepatol. 2017; 66(2): 282–287. PubMed Abstract | Publisher Full Text\n\nWorld Health Organization: HIV-HCV Overview.2017.[Accessed 2019].Reference Source\n\nAbiodun OE: OE-Abiodun/HIV-HCV-COINFECTION-SIM-CODE: HIV-HCV Co-infection (v3.0.0). Zenodo. [Source code]2022. Publisher Full Text"
}
|
[
{
"id": "153205",
"date": "08 Nov 2022",
"name": "Adewale F. Lukman",
"expertise": [
"Reviewer Expertise Biostatistics"
],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe idea presented in the article is innovative and well-written. I recommend acceptance for indexing subject to minor revision.\nThe authors developed and investigated a mathematical model for the dynamical behavior of HIV/AIDS and HCV co-infection, which includes therapy for both diseases, vertical transmission in HIV cases, unawareness and awareness of HIV infection, inefficient HIV treatment follow-up, and efficient condom use. The Methods and theorem in the articles are well stated and clear enough for future work purposes.\nThe article recommends that HCV should be treated first in those co-infected with HIV. This is a strong recommendation for the public health sectors to take note of in reducing the increasing rate of viral load in HIV patients. The authors should consider adding those inferred steps to the conclusion part of the abstract session for easy access for the reader.\nThe problem is addressed with a detailed numerical simulation. Conclusively, the authors should consider extending the work by adding controls on the model to know what control parameters will be recommended to control the menace of the co-infection.\nThe authors should go through the grammatical structure of the manuscript. For instance, I made a few revisions to the Abstract Background: \"Hepatitis C Virus (HCV) and Human Immunodeficiency Virus (HIV) are classified as blood-borne viruses since they are transmittable through contact with contaminated blood. Approximately 1.3 million of the 2.75 million global HIV/HCV carriers inject drugs (PWID). HIV co-infection harms the progression of HCV, resulting in higher rates of HCV persistence after acute infection, higher viral levels, and accelerated progression of liver fibrosis and end-stage liver disease.\"\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\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": "153204",
"date": "21 Nov 2022",
"name": "Afeez Abidemi",
"expertise": [
"Reviewer Expertise Mathematical modelling",
"mathematical biology",
"optimal control theory",
"nonlinear dynamics",
"population dynamics"
],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nIn this paper, the authors developed and rigorously analysed a compartmental mathematical model describing the dynamics of Human Immunodeficiency Virus (HIV) and Hepatitis C Virus (HCV) co-infection. I have the following comments/observations and questions on the paper:\nThe overall command of English seems good, however there are several grammatical errors all over the paper. For instance, in the abstract, “Hepatitis C Virus (HCV) and Human Immunodeficiency Virus (HIV) are both as classified blood-borne viruses since they are transmitted through contact with contaminated blood” is not grammatically correct. There are many incomplete sentences as well. For example, the phrase “and antiretroviral drugs are more harmful to the.” as appeared in the seventh line of the second paragraph under introduction section is incomplete. It is difficult to read the meaning of some sentences. An example is the sentence “Examining prevention, diagnosis, screening, HIV knowledge and awareness, condom use, and largely using numerical simulations, ignorance and awareness, and condom use and mostly employs numerical simulations”. Therefore, it is strongly advised and it is the authors’ responsibility to recheck the whole manuscript and fix all the grammatical errors. Moreover, in the entire manuscript, insert (,) and (.) wherever required in the equations.\n\nThere are no definitions for some acronyms; PLHIV, HBV, ODE, HAART, ART. The authors should ensure that they include the meaning of each of the acronyms at its first mentioning.\n\nUnder full model description, notations for the AIDS populace aware and on treatment and HIV-positive individuals in stage-IV co-infected with chronic HCV as given in the description (AA(t), AAC(t)) are different from what later appeared in the system Eq. (1) (Aa (t), AaC (t)). The authors should correct this. It is difficult to translate the flow diagram into the model equations in system (4). The authors should improve on the quality of the flow diagram of the proposed model.\n\nThe notation for AIDS-induced death rate given before Equation (2) does not align with the used notation (da) in the model equations. Please, correct it.\n\n“(ri, i = 1, 2, ...)” should not be enclosed in a bracket. More so, is index i uncountable? Please be specific about all the integer-indexed rates ri, θi, δi throughout the manuscript. For instance, “ ri, i = 1, 2, ...” should be ri, i = 1, 2, ..., 5 according to system (4). In Table 1, α1 should be αi. The statement “Mathematically, the flow chart leads to the 15 systems of ordinary differential equations listed below:” immediately before model (4) should be “Mathematically, the flow chart leads to the system of 15 ordinary differential equations given by”.\n\n“the system dynamics (3.4)” immediately before Eq. (6) should be “the system dynamics (4)”.\n\nIt is wrong to say that \"The system variable (1) are positive ...\" in Theorem 1 because (1) refers to the total population. Please, correct this accordingly.\n\nIn the proof of Theorem 1, the statement “It follows from the model's first equation (1) that” is misleading. The authors should make the right equation reference. In general, the authors should check the proof of Theorem 1. There are many mistakes. For instance, what is the meaning of “d/dt = ...”? The exponents are wrongly written. Take for instance, \"e[]\" should be \"e[]\". Lastly, how the authors arrived at the conclusion S ≥ 0 from the preceding expression is not clear. Please explain. What are R0' and RN as mentioned immediately under section \"Disease-free equilibrium and the effective reproduction number\"? Please, give their meanings.\n\nDFE of the full model (4) is E0HC not EoC. Correct accordingly.\n\nAccording to the authors, Theorem 2 should be Lemma 2. Please, make correction appropriately.\n\nWhat is “E c” in Theorem 3?\n\nWhat are the variables x1, x2, x3 that appear in the Jacobian matrix (16)? Are the trace < 0 and determinant > 0 always true for the Jacobian matrix? The authors should mention the conditions under which the trace is non-positive and determinant is positive.\n\nThe statement “… and the right side of the second equation is differentiated with regards to CC” immediately before Eq. (19) should be “… and the right side of the third equation is differentiated with regards to CC”. What is the physical interpretation of the result in Eq. (22)? The authors should add this result.\n\nWhat about the endemic equilibrium of the full system (4)? The authors should say something about it.\n\nThere is no section 2.2.1 as mentioned immediately before Eq. (23). The authors should correct this.\n\nIn Lemma 5, “submodel (9)” should be “submodel (12)”. Please, ensure this correction for the rest of this part.\n\nDisease-free equilibrium E0 is undefined throughout the manuscript. The authors should fix this.\n\n“GSA” as mentioned immediately before the numerical simulation section should be “GAS”.\n\nSection 2.2.3 in the caption of Figure 4, Theorem 3.12 in the caption of Figure 5.\n\nIn “conclusion section”, the last sentence should be merged with the paragraph before the paragraph that highlights the direction of future studies.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\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": "9058",
"date": "06 Jan 2023",
"name": "Oluwakemi Abiodun",
"role": "Author Response",
"response": "Dear Reviewer, Dr Afeez Abidemi. Thank you for your comments and recommendations. All comments, observations and recommendations from comment number 1 to 23 have been addressed. For comment number 11, R0 is the reproduction number while the RN is a typo which has been deleted. For comment number 14, Ec* is the endemic equilibrium point for the Hepatitis C virus (HCV) In comment 15, xi, x2, x3 are representation of the state variables Furthermore, for trace < 0 and determinant > 0, they are not always true for the Jacobian matrix. They are true under the condition for DFE and Endemic equilibrium (EE) whenever R0 < 1 and if R0 > 1 respectively. Physical interpretation of Eqn (22) now (23) mentioned in comment 17 has been included in the new version submitted. Thank you."
}
]
}
] | 1
|
https://f1000research.com/articles/11-1153
|
https://f1000research.com/articles/11-57/v1
|
18 Jan 22
|
{
"type": "Research Article",
"title": "Driving style recognition using machine learning and smartphones",
"authors": [
"Eilham Hakimie bin Jamal Mohd Lokman",
"Vik Tor Goh",
"Timothy Tzen Vun Yap",
"Hu Ng",
"Eilham Hakimie bin Jamal Mohd Lokman",
"Timothy Tzen Vun Yap",
"Hu Ng"
],
"abstract": "Background: The lack of real-time monitoring is one of the reasons for the lack of awareness among drivers of their dangerous driving behavior. This work aims to develop a driver profiling system where a smartphone’s built-in sensors are used alongside machine learning algorithms to classify different driving behaviors. Methods: We attempt to determine the optimal combination of smartphone sensors such as accelerometer, gyroscope, and GPS in order to develop an accurate machine learning algorithm capable of identifying different driving events (e.g. turning, accelerating, or braking). Results: In our preliminary studies, we encountered some difficulties in obtaining consistent driving events, which had the potential to add “noise” to the observations, thus reducing the accuracy of the classification. However, after some pre-processing, which included manual elimination of extraneous and erroneous events, and with the use of the Convolutional Neural Networks (CNN), we have been able to distinguish different driving events with an accuracy of about 95%. Conclusions: Based on the results of preliminary studies, we have determined that proposed approach is effective in classifying different driving events, which in turn will allow us to determine driver’s driving behavior.",
"keywords": [
"Machine learning",
"driver profiling",
"smartphone",
"convolutional neural networks"
],
"content": "Introduction\n\nDriver behavior strongly influences road safety1 and is currently the main contributor to traffic fatalities. Although many recorded incidents are caused by human errors, researchers suggest that drivers who exhibit a more aggressive driving style are more likely to be engaged in an accident on the road2. In 2018, a total of 548,598 cases of road accidents have been recorded in Malaysia with 6,284 of them resulting in fatalities3. To add to that, the Malaysian Institute of Road Safety Research Institute (MIROS) predicts that the number of fatalities will continue to increase up to 10,716 by the end of 20204. Driver profiling attempts to understand and monitor the driver’s behaviour in real-time, leveraging a safer and more responsible driving.\n\nDriving style profiling is the process of collecting driving data (e.g., acceleration, braking, speed, turning rate, location) then applying them to classification models in order to generate a score to determine whether their driving style is safe or unsafe. Driver profiling has gained an increased in demand particularly in the insurance field and rental fleets. For example, AXA FlexiDrive rewards their customers with up to a 20% discount off their insurance premiums if they display good driving behaviour5. In the rental car industry, rental companies turn to RentalMatics, an IoT solution to track rental fleets in real time6. A telematics unit (also called black box) is fitted into a car to gather the relevant data needed to monitor and track the vehicle’s location and behaviour7.\n\nToday’s smartphone is embedded with advanced motion sensors which is suitable for data collection. Smartphones have access to a suite of advanced sensors including accelerometer, gyroscope, magnetometer, gravity sensors and GPS. Previous works like8–11 proved that if smartphones are properly calibrated, they could be viable alternatives to the conventional telematics unit for monitoring driver behaviour.\n\nThis research aims to create a viable system to gather data through a mobile device and apply machine learning algorithms to classify different types of driving events. The research will explore the data gathering phase, where a smartphone will be used as a data collection device to replace the telematics device. Initial calibration of the motion sensors will be applied to ensure the most accurate reading of data for any vehicle. Machine learning algorithms will then be trained and tested to ensure the accuracy in determining different driving events such as careful, normal, careless, and dangerous.\n\n\nMethods\n\nThis project was divided into two main parts; the first part was the data collection part which is to develop an intuitive smartphone application with the correct combination of sensors to gather raw data on basic vehicular movements and the second part is applying machine learning to train and classify different driving patterns.\n\nData collection utilized the smartphone’s sensors including accelerometer, gyroscope, and GPS. The accelerometer’s readings (x, y and z axes) relative to the phone position were recorded every 100ms for the duration of the data collection. The gyroscope sensor was used to measure the vehicle’s rate of rotation while the GPS was used to calculate the speed of the vehicle. Figure 1 shows the user interface of the smartphone app. The app recorded several driving event such as:\n\nRight turns (90°)\n\nLeft turns (90°)\n\nAcceleration\n\nBraking\n\nThe driving events were attempted at a constant speed of 30km/h. For each driving event, 30 sets of readings were collected in total.\n\nThe figure shows the user interface of the data gathering app where readings from the smartphone’s accelerometer and gyroscope are updated in real-time.\n\nThe classification technique employed to assess the implementation of this approach was the 1-Dimensional Convolutional Neural Network (CNN) and the CNN model was built using Tensorflow and Keras. Prior to fitting the machine learning model with data, the data was pre-processed beforehand to overcome problems such as unbalanced data or redundant data which could cause overfitting of model.\n\nPre-processing began by balancing the data to ensure that the data points for all driving events are equal to prevent skewing of results. Data standardization was also implemented before model fitting. This step is vital to ensure that the model can handle lower valued numbers as the data from the sensors has fluctuations which would cause the result to be inaccurate and require more processing to compensate. Data standardization ‘minimizes’ the amplitude of the data into a form that is much easier to handle by the computer. Frame preparation was done during the pre-processing stage where the frame size was selected to be four seconds and the hop size was chosen to have an overlap of 24 data points. A total of 240 frames were created to accommodate the sample size. The sample data was then split into 80% for training set and 20% testing set.\n\nAfter pre-processing, the CNN layer was built. The neural network layer was built following multiple trial and errors to ensure the outcome produces a good-fit model graph. An initial 1-dimensional CNN layer called ‘Conv1D’ was created. The layer develops a convolutional kernel that convolves with the layer input over a single dimensional space to produce a tensor of outputs. Following the initial layer, an activation layer called the Rectified Linear Unit (ReLU) was applied as it is the ideal activation layer for Multilayer Perceptron (MLP) and CNN. ReLU is an activation function that utilizes simple arithmetic that will output the value directly if it is more than 0.0, and will output 0.0 if the input is less than 0.012. After that, a pooling layer by the name of ‘MaxPool1D’ was applied to reduce the number of feature maps by taking the maximum value over a certain pool size. This layer is recommended in CNN models as it reduces variance and minimizes computations13. A ‘Dropout’ layer is then applied which operates by setting the outgoing edges of each node to zero to minimize overfitting14. A ‘Dense’ layer is applied to allow the neurons in the ‘Dense’ layer to receive input from neurons of the previous layer15. Finally, a ‘Softmax’ layer is applied to convert the output into a probability distribution. Figure 2 visualizes the 1-dimension CNN used for this research.\n\nThe illustration above shows the various layers used to develop the machine learning model. The various layers include Conv1D layers, Rectified Linear Unit (ReLU) Activation function, Pooling layers, Dropout, and Dense layers.\n\n\nResults and discussion\n\nThe readings from the smartphone’s accelerometer for the various driving events are shown in Figure 3. Due to the vibrations of the vehicle during idling and during movements, a few spikes in the amplitude can be seen. The spikes in the accelerometer readings during idling could also be caused by the vehicle engine running.\n\n(a) The x-axis of the accelerometer form peaks at each Right turning. (b) Left turn creates an opposite pattern from Right turns as each turning point forms successive valleys upon the x-axis. (c) Accelerate event creates valleys on the z-axis of the accelerometer. (d) Brake event indicates an opposite pattern where peaks are formed on the z-axis of the accelerometer at each braking point.\n\nThe different types of driving events that were demonstrated could be easily distinguished between each other. In Figure 3(a), the accelerometer readings for the x-axis displays multiple peaks successively. Each peak in the graph translates to a right turn where the highest point of each peak translates to the highest force during each turn.\n\nOn the other hand, the readings for left turn events show the opposite result where in Figure 3(b), the accelerometer readings for the x-axis displays multiple valleys successively. Each valley translates to a single left turn in real life. However, Figure 3(a) and 3(b) display a close resemblance in the y and z-axis during the early phase of the recording, where the y and z-axis shows multiple peaks followed by multiple valleys. This could possibly be caused by the vibrations coming out from the engine of the car moving from a stationary position.\n\nIn Figure 3(c), the accelerometer values show readings for the vehicle during acceleration. A series of valleys are formed in the z-axis where each valley translates to an acceleration event being performed. The intensity of the valley is not very pronounced as the throttle was gradually applied to ensure smooth acceleration. If the accelerator was pressed more aggressively, we predict the z-axis value of the accelerometer to be more pronounced. Figure 3(d) shows the accelerometer readings for the vehicle under braking events. The output is the opposite of acceleration event, as the z-axis of the accelerometer form peaks at each point of braking. Each peak is much more visually defined as the brakes were applied much more aggressively to fully stop the car from 30km/h. In both Figure 3(c) and 3(d), the x and y-axis produced a few peaks followed by valleys. This could be caused by the inertia acting on the vehicle or the engine vibrations in each driving event.\n\nAs observed from the accelerometer plots, each driving event could be distinguished from each other as each event has their specific features. For left and right turns there is a distinct difference in their x-axes while acceleration and braking events can be distinguished in the z-axis. The difference in features for each driving event could be highly advantageous as we could train machine learning algorithms to identify their specific features and automatically categorize each of the driving events.\n\nThe machine learning experiment was carried in two phases, training phase and test phase. 80% of the total sample data was used to train the 1-CNN model and 20% of the sample data was used to test the accuracy of the trained model. After numerous experiments, it is discovered that the most optimal epoch value is 100 combined with a batch size of 32 which produced a model accuracy of 95.83%. Figure 4 shows the test results of the machine learning model16.\n\nThe figure visualizes the evaluation results for the model when classifying the testing set where an accuracy of 95.83% was obtained.\n\nThe Model Loss Curve in Figure 5 shows that the training loss decreases as the epoch value increases. Upon reaching epoch value 80, the training loss stops decreasing and stabilizes. Similarly, the validation loss decreases as the epoch value increases but stabilizes at around 80 epochs. It can be observed that the validation loss and training loss has very minimal gap between them which states that the model is a good fit model. Continued training of this model will likely cause the model to be overfitted.\n\nThe figure depicts the learning curves for Model Accuracy and Model Loss where each graph has a curve for Training and Validation performances.\n\nFigure 6 visualizes that out of the model predicted 46 out of 48 events correctly. The two events that were wrongly predicted was an Accelerate event which was supposed to be a Left turn event and a Braking event which was supposed to be an Accelerate event. A probable reason as to why the model predicted these events wrongly is because of high variance of data because of the difficulty in maintaining consistency in simulating the driving events. Additional pre-processing of data could also improve the accuracy of the model as it removes redundant data and noise. Increasing the epoch value could also further improve the accuracy but caution must be kept in mind to make sure that the model is not overfitted.\n\nThe figure shows the Confusion Matrix where the prediction results are summarized to show the performance of the model.\n\nAdditional work could be done to improve the quality of data samples. For example, investing on a phone mount that does not wobble too much under engine vibrations. The data gathering framework could also be improved to ensure that only the relevant data samples are collected and not redundant data. For the application side, the sampling rate could also be tweaked to ensure that redundant accelerometer and gyroscope readings are not recorded.\n\n\nConclusion\n\nSmartphone sensors have improved massively over the years and prove to be a viable option when used as a data gathering apparatus to monitor driver behavior. In turn, the collected data could then be processed using machine learning algorithms to classify different driving events with high reliability. For this research we have applied the concept of Convolutional Neural Networks which works effectively in classifying different driving events with an accuracy of 95.83%.\n\nFor our future work, we will continue to collect data for different driving events such as aggressive driving and careless driving. Additionally, we will feed the collected data into the machine learning model to allow it to classify an even more diverse palette of driving events. Next, we will consider adding a calibration feature into our data gathering application to allow accurate data collection without mounting the phone to a phone mount which will further improve user experience. As there are many machine learning models suitable for classifying time-series sensor data, we will experiment with different types of machine learning models to find out which will provide the best accuracy. Finally, we will deploy the machine learning model onto a smartphone to allow real-time classification of driving style. With these steps planned, it could potentially enhance the impact of this research in terms of traffic safety which was the main goal of this paper.\n\n\nData Availability\n\nHarvard Dataverse: Driving Events, https://doi.org/10.7910/DVN/F5JZHF16.\n\nThis project contains the following underlying data:\n\n- driving_events.db (Raw data from sensors)\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\nAbojaradeh M, Jrew B, Al-Ababsah H, et al.: The Effect of Driver Behavior on Freeway Traffic Flow. Civ Environ Res. 2014; 6(1): 39–54. Reference Source\n\nRoslin EN, Azmy NSA, Ghulam AM, et al.: Factors of the ‘aggressive driving’ behaviour amongst Malaysian drivers. Int J Eng Adv Technol. 2019; 8(6): 3359–3366. Publisher Full Text\n\nD. of Statistics: Department of Statistics Malaysia. Press Release, 2015.\n\nRohayu S: Predicting Malaysian Road Fatalities for Year 2020. 2020. Reference Source\n\nWhat You Need To Know About Telematics Motor Insurance. AXA Malaysia, (accessed Dec. 14, 2020). Reference Source\n\nTelematics For Rental Fleets: Shaping the Mobility Industry. RentalMatics, (accessed Dec. 14, 2020). Reference Source\n\nTelematics Explained - What is Telematics? (accessed Dec. 14, 2020). Reference Source\n\nJúnior JF, Carvalho E, Ferreira BV, et al.: Driver behavior profiling: An investigation with different smartphone sensors and machine learning. PLoS One. 2017; 12(4): e0174959. PubMed Abstract | Publisher Full Text | Free Full Text\n\nFazeen M, Gozick B, Dantu R, et al.: Safe Driving Using Mobile Phones. IEEE Trans Intell Transp Syst. 2012; 13(3): 1462–1468. Publisher Full Text\n\nCastignani G, Derrmann T, Frank R, et al.: Driver behavior profiling using smartphones: A low-cost platform for driver monitoring. IEEE Intell Transp Syst Mag. 2015; 7(1): 91–102. Publisher Full Text\n\nJohnson DA, Trivedi MM: Driving style recognition using a smartphone as a sensor platform. IEEE Conf Intell Transp Syst Proceedings ITSC. 2011; 1609–1615. Publisher Full Text\n\nA Gentle Introduction to the Rectified Linear Unit (ReLU). (accessed Jun. 22, 2021). Reference Source\n\ntf.keras.layers.MaxPool1D. TensorFlow Core v2.5.0., (accessed Jun. 22, 2021). Reference Source\n\nDropout Neural Network Layer In Keras Explained. by Cory Maklin | Towards Data Science, (accessed Jun. 22, 2021). Reference Source\n\nKeras Dense Layer Explained for Beginners. MLK - Machine Learning Knowledge, (accessed Jun. 22, 2021). Reference Source\n\nGoh VT, Lokman JM, Hakimie E, et al.: Driving Events. Harvard Dataverse, 2021. http://www.doi.org/10.7910/DVN/F5JZHF"
}
|
[
{
"id": "122549",
"date": "28 Feb 2022",
"name": "Muhammad Reza Z'aba",
"expertise": [
"Reviewer Expertise Information security",
"cryptography"
],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe article describes an attempt to use mobile phone as an apparatus to collect data regarding certain driving events such as turning, accelerating or braking. Then, convolutional neural network (CNN) is applied to classify these different events. The authors claim that CNN managed to classify the events with a high accuracy (95.83%).\nI think the title is a bit misleading. The title mentions about driving style recognition. However, what the work recognises was the driving events such as turning, accelerating or braking. When \"style\" was mentioned, I was thinking something along the lines of \"careless driving\" or \"careful driving\". In essence, I believe the work has yet to recognise the driving style, only the driving events. It is good if the authors can categorise several driving styles according to a set of driving events.\nThe authors mention that in order to improve data collection, it is proposed to use a phone mount in order to eliminate inaccurate readings due to engine vibrations. I think the authors should do this and include the results in the article since I think a phone mount is not that expensive and is widely available. The results can then perhaps be compared with the data collected without using the phone mount. This is to see how much accuracy can be gained.\nThe authors also did not state the model of the smartphone used in the work. It is also good if different smartphone models are used in order to make a comparison.\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Partly\n\nAre sufficient details of methods and analysis provided to allow replication by others? Partly\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nNot applicable\n\nAre all the source data underlying the results available to ensure full reproducibility? Partly\n\nAre the conclusions drawn adequately supported by the results? Partly",
"responses": [
{
"c_id": "9129",
"date": "19 Dec 2022",
"name": "Vik Tor Goh",
"role": "Author Response",
"response": "The title will be revised to “Driving event recognition using machine learning and smartphones” to better reflect the content of the paper. However, this is subject to the approval of F1000’s editors. A phone mount was indeed used to collect data in the current experimental setup. The mount was semi-rigid as this provided a good transfer of energy/vibration from the vehicle as well as some dampening against minor vibrations. It functioned as intended but also introduced some unwanted wobble due to its semi-rigid build. As such, in our future experiments, we will investigate using other models of mounts with different build quality. The experiments used a Samsung Galaxy S10 as the data collector. The paper has been revised to include some additional information about the data collection setup. A comparison of different smartphone models was not in the scope of the current phase but will be considered in our future experiments."
}
]
},
{
"id": "143359",
"date": "08 Jul 2022",
"name": "Babul Salam KSM Kader Ibrahim",
"expertise": [
"Reviewer Expertise Sensors",
"Modelling and Control"
],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis paper discusses the attempt to use the smartphone sensors such as accelerometer, gyroscope, and GPS in order to develop an accurate machine learning algorithm capable of identifying different driving events (e.g. turning, accelerating, or braking). The convolutional neural network (CNN) has been applied to classify these different events.\n\nThe title should be corrected as the work recognises the driving events, not the driving style.\n\nThe methodology should be improved by explaining and showing where the smartphone is mounted on the screen or on the dashboard. The illustration may help the reader.\n\nMounting on the dashboard is not a good option as there are vibrations due to the car’s engine. This should be avoided.\n\nThe labels in Figure 3 are not very clear.\n\nPlease proofread the document before submission. There are too many grammatical mistakes and need to be addressed as follows-\nReplace Conclusions: Based on the results of preliminary studies, we have determined that the proposed approach is effective in classifying different driving events, which in turn will allow us to determine driver’s driving behavior. with Conclusions: Based on the results of preliminary studies, we have determined that the proposed approach is effective in classifying different driving events, which in turn will allow us to determine driver’s driving behavior.\nReplace Driver profiling attempts to understand and monitor the driver’s behaviour in real-time, leveraging a safer and more responsible driving. with Driver profiling attempts to understand and monitor the driver’s behaviour in real-time, leveraging safer and more responsible driving.\n\nReplace The app recorded several driving event such as: with The app recorded several driving events such as:\nReplace Prior to fitting the machine learning model with data, the data was pre-processed beforehand to overcome problems such as unbalanced data or redundant data which could cause overfitting of model. with Prior to fitting the machine learning model with data, the data was pre-processed beforehand to overcome problems such as unbalanced data or redundant data which could cause overfitting of the model.\nReplace The neural network layer was built following multiple trial and errors to ensure the outcome produces a good-fit model graph. with The neural network layer was built following multiple trials and errors to ensure the outcome produces a good-fit model graph.\nReplace Figure 3. Accelerometer readings. (a) The x-axis of the accelerometer form peaks at each Right turning. (b) Left turn creates an opposite pattern from Right turns as each turning point forms successive valleys upon the x-axis. (c) Accelerate event creates valleys on the z-axis of the accelerometer. (d) Brake event indicates an opposite pattern where peaks are formed on the z-axis of the accelerometer at each braking point. with Figure 3. Accelerometer readings. (a) The x-axis of the accelerometer form peaks at each Right turn. (b) The Left turn creates an opposite pattern from Right turns as each turning point forms successive valleys upon the x-axis. (c) Accelerate event creates valleys on the z-axis of the accelerometer. (d) The brake event indicates an opposite pattern where peaks are formed on the z-axis of the accelerometer at each braking point.\nReplace Figure 3(a) and 3(b) display a close resemblance in the y and z-axis during the early phase of the recording, where the y and z-axis shows multiple peaks followed by multiple valleys. with Figures 3(a) and 3(b) display a close resemblance in the y and z-axis during the early phase of the recording, where the y and z-axis show multiple peaks followed by multiple valleys.\nReplace\nAs observed from the accelerometer plots, each driving event could be distinguished from each other as each event has their specific features. with As observed from the accelerometer plots, each driving event could be distinguished from the other as each event has its their specific features.\nReplace The machine learning experiment was carried in two phases, the training phase, and test phase with The machine learning experiment was carried out in two phases, the training phase and the test phase\nReplace A probable reason as to why the model predicted these events wrongly is because of high variance of data because of the difficulty in maintaining consistency in simulating the driving events. with A probable reason why the model predicted these events wrongly is because of the high variance of data because of the difficulty in maintaining consistency in simulating the driving events.\nReplace It can be observed that the validation loss and training loss has very minimal gap between them which states that the model is a good fit model with It can be observed that the validation loss and training loss have a very minimal gap between them which states that the model is a good fit model\nReplace Figure 6 visualizes that out of the model predicted 46 out of 48 events correctly. with Figure 6 visualizes that the model predicted 46 out of 48 events correctly.\n\nReplace Next, we will consider adding a calibration feature into our data gathering application to allow accurate data collection without mounting the phone to a phone mount which will further improve user experience. with Next, we will consider adding a calibration feature to our data gathering application to allow accurate data collection without mounting the phone to a phone mount which will further improve the user experience.\n\nIs the work 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": "9130",
"date": "19 Dec 2022",
"name": "Vik Tor Goh",
"role": "Author Response",
"response": "The title will be revised to “Driving Event recognition using machine learning and smartphones” to better reflect the content of the paper. However, this is subject to the approval of F1000’s editors. The smartphone was placed on the center dashboard via a phone mount. The paper has been revised to include this information for better clarity. We have already tested different position such as left side of the dashboard, right side of the dashboard, and even on the center console below the radio but came to a conclusion that the center of the dashboard was the best option for this car. The labels in Figure 3 have been enhanced for better clarity. All suggested amendments have been done. The rest of the document has also been proofread to the best of our ability to minimise grammatical and typological errors. Thank you very much for highlighting the errors."
}
]
}
] | 1
|
https://f1000research.com/articles/11-57
|
https://f1000research.com/articles/11-1533/v1
|
19 Dec 22
|
{
"type": "Research Article",
"title": "How adjusting elicited health utilities after the fact can adversely affect shared decision making",
"authors": [
"Edouard Kujawski"
],
"abstract": "Background: The elicitation of inconsistent health-state utility values (HSUVs) is a prevalent problem. There are two approaches to address this problem: (1) intervention during the elicitation process to ensure that patients estimate consistent HSUVs; (2) no intervention during the elicitation process and inconsistent HSUVs are adjusted after the fact. This paper studies three models recently proposed for adjusting inconsistent HSUVs and consistent HSUVs that some may consider unrealistic. Analysis: The three models are analyzed using a sound theoretical framework: the mathematical equivalence of HSUVs elicited using the standard gamble and probabilities, the Fréchet bounds, and preference theory. It is proven that none of these models accounts for the Fréchet lower bound and health conditions that are preference substitutes. Results: A clinical vignette proves these models may recommend treatments that result in premature death over treatments that cause acceptable adverse effects. Conclusions: The three models are incorrect and may mislead patients and physicians to poor medical decisions. In the spirit of shared decision making, patients should be given the opportunity to reassess inconsistent HSUVs and confirm that the revised HSUVs reflect their preferences.",
"keywords": [
"Elicitation intervention",
"Fréchet bounds",
"Health-state utility values",
"Preference complements",
"Preference substitutes",
"Reasonableness test"
],
"content": "Introduction\n\nThe elicitation of inconsistent health-state utility values (HSUVs) is a prevalent problem. A rational person should not prefer a joint health state (JHS) to any of the constituent single health states (SHSs). This property is termed logical consistency.1 For instance, at the individual level, 41% of the HSUVs for the JHS (incontinence & impotence) elicited by Dale et al.1 violated this property. Given the mathematical equivalence of HSUVs elicited using the standard gamble (SG) and probabilities, logical consistency corresponds to the Fréchet upper bound (FUB).2 It is highly likely that the percentage of inconsistent HSUVs was higher than 41% because Dale et al. did not consider the Fréchet lower bound (FLB).2 The later has significant implications for the disutility of multiple coexisting morbidities: the joint disutility cannot exceed immediate death (ID) or the sum of the individual disutilities.2\n\nTwo approaches to the problem of eliciting inconsistent HSUVs have been proposed:\n\n1. Prevent inconsistent elicited HSUVs through interviewers intervening whenever necessary to ensure that patients estimate consistent HSUVs.3\n\n2. No intervention during the elicitation process. Inconsistent elicited HSUVs are adjusted after the fact.4,5\n\nThe first approach requires trained and knowledgeable interviewers. The second approach requires realistic and mathematically valid models. However, the adjusted HSUVs may not accurately represent a patient’s preferences.\n\nTriantaphyllou and Yanase4 (referred to as T-Y in this paper) proposed three models for adjusting inconsistent as well as consistent HSUVs which they say “may still not be realistic”:\n\n– Model (i): “Readjusting the original health utility values via an error minimization approach based on the monotonicity property.”\n\n– Model (ii): “Multiplicative functions for health states and a new model for adjusting the initial health-state utilities.”\n\n– Model (iii): “A combined approach for adjusting the initial health-state utilities.”\n\nThis study has a sound theoretical framework: the mathematical equivalence of HSUVs elicited using SG and probabilities.2 It uses probability theory and preference theory to prove that the T-Y models are incorrect. A simple clinical vignette (Box 1) demonstrates that these models can be misleading. Therefore, they are inappropriate for shared decision making (SDM) where reliable HSUVs are critical for patients and physicians to decide upon a preferred treatment.6\n\nAt their annual physical examination, patient HP is diagnosed to suffer from the asymptomatic health condition X. HP is otherwise in good health. HP is neither a trained nor innate probability assessor.\n\nThe physician is a proponent of SDM, and HP agrees to participate.\n\nThe physician informs HP that:\n\n– X would reduce their expected years of life from 15 to 7, unless treated\n\n– Treatment Tx has a 100% success rate in curing X\n\n– Tx has a 100% probability of two side effects: ai and bj.\n\n– HP agrees to partake in assessing the HSUVs for ai, bj and ai&bj.\n\nThis paper proceeds as follows. The theoretical framework used for analyzing the T-Y models is presented. These models are analyzed, and it is demonstrated that they are inappropriate for life-critical SDM. Concluding remarks are presented.\n\n\nMethods\n\nHealth-state utility values\n\nHealth states are identified by health conditions (HCs) (also termed “attributes” and “dimensions”) and severity levels. Each combination of levels of HCs represents a unique health state. HSUVs measure the strength of a person’s subjective preferences for health-related quality of life (HRQL).7 They are cardinal values specified on the (ID = 0.0, perfect health (PH) = 1.0) scale.\n\nEstimating HSUVs is challenging. People are affected by the information which they receive, emotional factors, and elicitation methods.7 HSUVs elicited by different methods may not agree and can affect treatment choices.8 The SG has a theoretical foundation in von Neumann-Morgenstern expected utility theory9, which establishes the validity of HSUVs elicited using the SG as a measure for HRQL.10 Using arbitrary scales for HSUVs can lead to serious errors.11,p.17\n\nProbabilities: Fréchet inequalities\n\nFréchet12 proved that the joint probability of two events is bounded by the marginal probabilities of each event regardless of the dependence between them. For two events A and B,\n\nFew people realize they assign inconsistent values to joint probabilities. Osherson et al.13 state: “It is striking to observe, for example, how few people realize that it is inconsistent to attribute probabilities of 0.8 to each of two sentences and probability 0.5 to their conjunction.” From (1), the conjunction of 0.8 and 0.8 cannot be less than 0.6: PFLB0.8∧0.8=0.8+0.8−1.0.\n\nMathematical equivalence of HSUVs and probabilities\n\nIn the SG, an individual is asked to make the hypothetical choice between living for T years with health stateai (health condition Awith severity level i) and a gamble with a binary outcome (probability p of living in PH for T years or ID with probability 1.0−p). The probability p is varied until the individual is indifferent between living T years with ai and the gamble. The indifference probability paicorresponds to the individual’s HSUV for ai:10 Uai=pai. Given the mathematical equivalence of HSUVs elicited using the SG and probabilities, probability theory can be applied to the problem of identifying inconsistent HSUVs.2\n\nConsistent HSUVs: Fréchet inequalities\n\nGiven that HSUVs are mathematically equivalent to probabilities, the Fréchet inequalities play an important role in identifying inconsistent HSUVs. Uai&bj is bounded by the FUB and FLB on conjunction irrespective of preference interactions:2\n\nThe FUB (2b) ensures logical consistency.1 The FLB (2c) has significant implications for the disutility of multiple coexisting morbidities. The joint disutility cannot exceed ID or the sum of the individual disutilities:2\n\nPreference interactions\n\nHCs can be mutually utility independent (MUI), preference complements (PCs), or preference substitutes (PSs).2 If a patient’s preference for condition A is independent of the level of condition B and vice versa, A and B are said to be MUI: UMUIai&bj=Uai×Ubj. If a patient believes that both A and B need to improve for their HRQL to improve, A and are said to be PCs. PC HSUVs are positively correlated: UMUIai&bj<UPCai&bj≤FUB. If a person believes that only A or only B needs to improve for their HRQL to improve, A and B are PSs. PS HSUVs are negatively correlated: FLB≤UPSai&bj<UMUIai&bj.\n\nQuality-adjusted life-years\n\nThe linear quality-adjusted life-year (simply termed the QALY) is presently the principal model for medical decision making (MDM). The expected number of QALYs for living yi years in a health state xi which has a probability of occurrencepxi is10\n\n\nAnalysis of T-Y models\n\nIn the following subsections, the T-Y models4 are analyzed using the above theoretical framework and data shown in Table 1.14 The analysis was done using Microsoft Excel 2016.\n\nModel (i):4 “Readjusting the original health utility values via an error minimization approach based on the monotonicity property.”\n\nThe monotonicity property requires that a rational individual should not prefer a JHS to any of the constituent health states. Hence, Model (i) satisfies the FUB (2b). For consistency with probability theory, HSUVs elicited using the SG are also required to satisfy the FLB (2c). Model (i) does not address the FLB. For instance, it does not identify the HSUVs Uai=0.62,Ubj=0.73, Uai&bj=0.15 as inconsistent: Uai&bj<UFLB0.62∧0.73=0.35=0.62+0.73−1.0.\n\nModel (ii):4 “Multiplicative functions for health states and a new model for adjusting the initial health-state utilities.”\n\nModel (ii) posits Uiiai&bj=Uai×Ubj. This assumes that health conditions ai and bj are MUI.\n\nKeeney & Raiffa advocated assuming MUI with the significantly important qualifier15,p. 244: “the utility independence assumptions are appropriate in many realistic problems”. Moreover, their focus was principally on decision making outside of the medical domain. More recently, Howard and Abbas wrote16,p. 578\n\n“We have several issues with this type of ‘utility independence’ reasoning … Enforcing these ‘utility independence’ assumptions result in functional forms that are simple, but quite frequently they will not represent the preference of the decision maker.”\n\nExperimental studies have concluded that the multiplicative model is not a suitable model for JHSUVs.17 MUI is a strong assumption that is usually inappropriate for HSUVs.18\n\nModel (iii):4 “A combined approach for adjusting the initial health-state utilities”\n\nModel (iii) posits that JHSs have a level of utility independence controlled by a parameter 0.0≤γ≤0.1. Thus, the JHSUVs lie between the MUI HSUV and the FBU (2b) and they do not account for HCs that are PSs2. For instance, given Uai=0.62 and Ubj=0.73, Model (iii) predicts 0.45≤Uiiiai&bj≤0.62. This is wrong: HCs can be PSs, in which case 0.35≤UPSai&bj<0.45.\n\nT-Y4 recommend using Models (ii) and (iii) for JHSUVs that “would easily pass the previous monotonicity test but could still be considered as not realistic.” This can mislead clinicians to recommend and patients to choose unwanted treatments. Case in point, a patient who wants to avoid treatments with HSUVs ≤0.45 chooses treatment TX based on Uiiai&bj and Uiiiai&bj≥0.45.\n\n\nResults and discussion\n\nThe clinical vignette in Box 1 is analyzed assuming HSUVs that are elicited with and without intervention.\n\nInterviewers intervene when necessary to ensure that patient HP assesses consistent HSUVs which truly represent their preferences. Elicited single HSUVs (SHSUVs) are not always more correct than elicited JHSUVs.1 HP adjusts the JHSUV and SHSUVs as shown in Box 1: Uai=0.55, Ubj=0.62, Uai&bj=0.38. These HSUVs satisfy the FUB (= 0.55) and FLB (= 0.17).\n\nInterviewers do not intervene during the elicitation of HSUVs. The no-intervention elicited HSUVs shown in Table 1 violate the FLB (= 0.35). As discussed above, the T-Y models do not identify these HSUVs as inconsistent. T-Y recommend using Models (ii) and (iii) for JHSUVs that “could still be considered as not realistic.”4 These models predict the significantly different JHSUVs shown in Table 1.\n\nFor illustration, we consider the clinical vignette and data shown in Box 1 and Table 2, respectively. Patient HP has a complicated decision to make: “to be or not to be” treated with TX? The expected number of QALYs for each alternative and set of HSUVs is calculated using (4). Table 2 summarizes the results and recommendations. The HSUVs elicited with and without intervention provide contradictory recommendations:\n\n– HSUVs elicited with intervention. The prediction is: 15.0 YLs, 7.8 QALYs. The recommendation is “Yes Tx”.\n\n– HSUVs elicited without intervention and adjusted after the fact. Model (i) predicts 15 YLs and 2.25 QALYs. Models (ii) predicts 15 YLs and 6.75 QALYs. Based on the number of QALYs, Models (i,) and(ii) recommend “No Tx”. Model (iii) recommends either “No Tx” or “Yes Tx” depending on the control parameter γ.\n\nKujawski et al.19 proposed an intuitive reasonableness test that decision models used for SMD should pass to qualify as SDM tools: “Can a treatment that results in premature death trump a treatment that causes acceptable adverse effects?” A “Yes” answer may mislead clinicians into recommending and patients into choosing decisions with unintended consequences. As shown in Table 2, the three T-Y models fail this test.\n\n\nConclusions\n\nThe elicitation of reliable HSUVs is critical to ensure medical decisions that patients truly prefer. As shown in this paper, the three T-Y models4,5 do not accurately account for individual preferences and the mathematical equivalence of HSUVs with probabilities elicited using the SG. Given consistent elicited HSUVs, it is not the function of clinicians to judge whether these are realistic or unrealistic.\n\nA clinical vignette proves that the three T-Y models may recommend treatments that result in premature death over treatments that cause acceptable adverse effects. This is a sure sign that these models are faulty and can be misleading. Well-trained interviewers are still essential to elicit reliable HSUVs. Practical tools are being developed to assist with the assessment of HSUVs, e.g., Gambler II.20 The uncertainties of elicited HSUVs and calculated QALYs need to be addressed for sound SDM. Assuming point estimates causes false confidence in the analysis results.21,22",
"appendix": "Data availability\n\nFigshare: Elicited and adjusted HSUVs using T-Y models. https://doi.org/10.6084/m9.figshare.21651911. 14\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).\n\n\nReferences\n\nDale W, Biller P, Basu A, et al.: The prevalence, correlates, and impact of logically inconsistent preferences in utility assessments for joint health states in prostate cancer. Med. Care. 2011; 49(1): 59–66. PubMed Abstract | Publisher Full Text\n\nKujawski E: The importance of preference interactions in joint health-state utility values applied to decision analyses for shared decision-making. IISE Trans. Healthc. Syst. Eng. 2022; 1–14. Publisher Full Text\n\nStewart ST, Lenert L, Bhatnagar V, et al.: Utilities for prostate cancer health states in men aged 60 and older. Med. Care. 2005; 43(4): 347–355. Publisher Full Text\n\nTriantaphyllou E, Yanase J: How to identify and treat data inconsistencies when eliciting health-state utility values for patient-centered decision making. Artif. Intell. Med. 2020; 106: 101882. Publisher Full Text\n\nTriantaphyllou E, Yanase J: Treatment selection for life-critical shared decision making under ranges of health-state utility scenarios. J. Biomed. Inform. 2021; 115: 103604. Publisher Full Text\n\nLafata EJ, Brown RF, Pignone MP, et al.: Primary care physicians’ support of shared decision making for different cancer screening decisions. Med. Decis. Mak. 2017; 37(1): 70–78. PubMed Abstract | Publisher Full Text | Free Full Text\n\nGuyatt GH, Feeny DH, Patrick DL: Measuring health-related quality of life. Ann. Intern. Med. 1993; 118(8): 622–629. Publisher Full Text\n\nElkin EB, Cowen ME, Cahill D, et al.: Preference assessment method affects decision-analytic recommendations: a prostate cancer treatment example. Med. Decis. Mak. 2004; 24(5): 504–510. PubMed Abstract | Publisher Full Text\n\nvon Neumann J , Morgenstern O: Theory of games and economic behavior. Princeton University Press;1947.\n\nHunink MGM, Weinstein MC, Wittenberg E, et al.: Decision making in health and medicine: integrating evidence and values. Cambridge University Press; 2nd edition2014.\n\nAbbas AE: Foundations of multiattribute utility. Cambridge University Press;2018.\n\nFréchet M: Généralisations du théorème des probabilités totales. Fundam. Math. 1935; 25(1): 379–387. Publisher Full Text\n\nOsherson D, Lane D, Hartley P, et al.: Coherent probability from incoherent judgment. J. Exp. Psychol. Appl. 2001; 7(1): 3–12. PubMed Abstract | Publisher Full Text\n\nKujawski E: Elicited and adjusted HSUVs using T-Y models. figshare Dataset.2022. Publisher Full Text\n\nKeeney RL, Raiffa H: Decisions with multiple objectives: preferences and value tradeoffs. John Wiley & Sons;1976.\n\nHoward RA, Abbas AE: Foundations of decision analysis. Pearson;2015.\n\nFu AZ, Kattan MW: Utilities should not be multiplied: evidence from the preference-based scores in the United States. Med. Care. 2008; 46(9): 984–990. Publisher Full Text\n\nKujawski E: On the appropriateness/inappropriateness of the Keeney-Raiffa multiplicative utility function for medical decision making. PPI SyEN. 2019; 79: 5–17.Reference Source\n\nKujawski E, Triantaphyllou E, Yanase J: Additive multicriteria decision analysis models: misleading aids for life-critical shared decision making. Med. Decis. Mak. 2019; 39(4): 437–449. PubMed Abstract | Publisher Full Text\n\nBehney AC: Ignoring uncertainty in predictor variables leads to false confidence in results: a case study of duck habitat use. Ecosphere. 2020; 11(10): 1–13.\n\nAdejare AA Jr, Eckman MH: Automated tool for health utility assessments: the Gambler II. MDM Policy & Practice. 2020; 5(1): 1–12.\n\nSpiegelhalter DJ, Franklin RC, Bull K: Assessment, criticism and improvement of imprecise subjective probabilities for a medical expert system. Proceedings of the fifth workshop on uncertainty in artificial intelligence. 1989; 335–342."
}
|
[
{
"id": "235536",
"date": "14 Feb 2024",
"name": "Michal Lewandowski",
"expertise": [
"Reviewer Expertise Preference modelling",
"decision theory under risk",
"uncertainty",
"time delay",
"imprecision",
"fuzzy preferences."
],
"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\nReport on the article How adjusting elicited health utilities after the fact can adversely affect shared decision making, written by Eduard Kujawski and submitted to F1000.\nThe article discusses the consistency of utility values of health states. In particular, it is noted that the utility values obtained using a standard gamble are the probability equivalent of a gamble in which one can win perfect health or lose by immediately dying. This is the probability at which the decision maker is indifferent whether he will be in a given state of health for sure or whether he will take a risk with this probability of achieving perfect health; otherwise, instant death occurs. Therefore, the (dis-) utility of two individual states a, b (e.g. two side effects) occurring simultaneously must satisfy Frechet's probability inequality: it must be greater than max(0,pa+pb-1) and smaller than min(pa,pb). The point is, of course, right, but also quite obvious. To make a meaningful contribution, I would expect the paper to clearly show which existing models violate these consistency requirements. Of course, these do not include models that satisfy the mutual utility independence: since they assume independence, they clearly fall within Frechet's limits. However, the author states that these models are not realistic. The author discusses models (i) – (iii). However, they are not even properly defined, only a numerical example is presented. I think a publishable article should clearly define these models and discuss why they are important/where they are used etc. From the numerical example presented in the article, it can be concluded that model (i) does not satisfy Frechet's inequality (joint disutility/probability is less than Frechet's lower bound), model (ii) satisfies independence (but is called unrealistic), and model (iii) assumes that a and b are preference complements. Well, so what? The author states that none of the models is satisfactory because it may happen that a and b are preference substitutes and their joint probability falls somewhere between Frechet's lower bound and the product of two individual probabilities. The point, however, is that without additional knowledge we cannot say what it really is: whether a, b are preference substitutes, their complements, or whether they are independent. Frechet's inequalities must be explicitly satisfied if utility values are constructed as probabilities, this is clear. There are many things that can be criticized in this article - for example, the definition given by equation (4). However, since I believe that, for the reasons I have described above, the work is not suitable for publication, even if these defects are removed, I will not mention them and recommend rejection of the article.\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 source data required\n\nAre the conclusions drawn adequately supported by the results? No",
"responses": []
},
{
"id": "235544",
"date": "30 Oct 2024",
"name": "Pierfelice Cutrufelli",
"expertise": [
"Reviewer Expertise As a practicing psychiatrist",
"I limit this peer review to the clinical aspects of this manuscript",
"particularly those concerning shared decision-making and patient preference assessment. My expertise in mental health care",
"where patient preferences and treatment choices are uniquely complex",
"allows me to evaluate the practical implications of preference elicitation methods in clinical settings."
],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nAs a clinician, I will focus my peer review on the clinical aspects of this paper while acknowledging that I am not qualified to evaluate its mathematical components. I must first clarify that I am not qualified to assess the mathematical models, Fréchet bounds, or utility calculations presented in this paper. These aspects should be evaluated by experts in mathematical modeling and decision theory.\nClinical Relevance: The paper addresses an important clinical concern: how we measure and use patient preferences in shared decision-making (SDM). Recent work by Rodolico et al. (2023) examining SDM in psychiatric settings found that trust and decision-making involvement alone may not enhance patient empowerment, suggesting that preference elicitation is more complex than previously thought. This complexity makes the current paper's examination of health utility values particularly relevant to clinical practice, especially when discussing treatments with multiple potential side effects.\nClinical Vignette Assessment: The clinical vignette (Box 1) effectively illustrates a common scenario where:\nA patient has an asymptomatic condition requiring treatment The treatment has guaranteed side effects We need to help patients weigh longevity against quality of life\nWhile the vignette focuses on physical health outcomes, Rodolico's findings suggest we should also consider how patient empowerment and trust levels influence preference expression and stability.\nAs a clinician reviewing this paper, I strongly recommend a substantial expansion of the clinical components, particularly regarding Shared Decision Making (SDM). The current mathematical focus, while valuable, needs to be balanced with robust clinical context and practical guidance. Here are my detailed suggestions for enhancement. This is a comprehensive outline, and the author should decide what to focus on and what to write about. Not all parts need to be addressed:\n1. Comprehensive SDM Framework The paper would benefit from a dedicated section explaining:\na) Core SDM Components:\nDefinition and evolution of SDM in clinical practice Current best practices in SDM implementation Barriers and facilitators to effective SDM Role of patient empowerment, as highlighted by Rodolico et al. (2023) Integration of trust dynamics in the SDM process\nb) Clinical Context:\nReal-world applications across different medical specialties Impact of time constraints on SDM implementation Variation in SDM approaches based on clinical settings (acute vs. chronic care) Influence of symptom severity on decision-making capacity\n2. Expanded Clinical Considerations The paper should address: a) Patient Factors:\nHealth literacy levels Cultural and linguistic considerations Role of family and caregivers Impact of cognitive status Emotional state and its influence on preference expression Prior healthcare experiences\nb) Provider Factors:\nClinical expertise integration Time management strategies Communication skills requirements Documentation practices Risk communication approaches\n\nPractical Implementation Guidelines\nAdd sections on: a) Clinical Workflow Integration:\nStep-by-step guidance for preference elicitation Time-efficient approaches Documentation templates Quality assurance measures Follow-up protocols\nb) Training Requirements:\nClinician preparation needs Staff role definition Ongoing education requirements Competency assessment methods\n3. Clinical Vignette Expansion Current vignette should be expanded to include: a) Multiple Scenarios:\nVarying complexity levels Different clinical specialties Range of patient characteristics Various outcome possibilities\nb) Detailed Process Description:\nInitial patient engagement Information sharing methods Preference elicitation techniques Decision documentation Follow-up planning\n4. Quality Metrics and Outcomes Add discussion of: a) Clinical Outcomes:\nPatient satisfaction measures Treatment adherence rates Health outcomes correlation Quality of life impacts Long-term follow-up results\nb) Process Measures:\nTime efficiency metrics Resource utilization Cost implications Staff satisfaction\nSystem integration success\n5. Special Considerations Include sections on: a) Vulnerable Populations:\nElderly patients Mental health conditions Language barriers Cultural differences Limited health literacy\nb) Complex Clinical Situations:\nEmergency decision-making Chronic disease management End-of-life care Multiple comorbidities Conflicting treatment goals\n6. Integration with Existing Clinical Systems Address: a) Electronic Health Record Integration:\nDocumentation requirements Decision support tools Workflow optimization Data collection methods Quality monitoring\nb) Team-Based Care:\nRole definition Communication protocols Handoff procedures Collaborative decision-making Cross-disciplinary coordination\n7. Research Implications Expand on: a) Clinical Research Needs:\nOutcome studies design Implementation research Quality improvement metrics Patient-reported outcomes Long-term impact assessment\nb) Knowledge Gaps:\nCurrent limitations Future research directions Methodology improvements Validation requirements Translation to practice\nThese expansions would significantly strengthen the paper's clinical utility and provide a more balanced perspective between mathematical rigor and practical application. The enhanced clinical content would make the paper more accessible and valuable to healthcare providers while maintaining its theoretical contributions.\nClinical Concerns: The paper's key clinical message - that post-hoc adjustments to patient preferences could lead to recommendations for premature death over manageable side effects - is deeply concerning from a medical ethics perspective. This concern is amplified by Rodolico's finding that higher patient empowerment correlates with better clinical outcomes, suggesting that undermining patient preferences through post-hoc adjustments might adversely affect treatment outcomes.\nPractical Clinical Implications: As a clinician, I have reservations about:\nThe practicality of conducting detailed preference assessments in time-constrained clinical settings How to balance trust and active patient involvement, given Rodolico's finding that trust may paradoxically decrease patient engagement The impact on clinical workflow and patient comprehension How symptom severity might affect preference stability and consistency\nRecommendation: While I support publication from a clinical perspective due to its relevance to medical decision-making, I strongly recommend: a) Additional peer review by mathematical experts for the theoretical framework b) Clear separation of clinical and mathematical components in the paper c) More practical guidance for implementing preference elicitation in clinical settings d) Integration of recent findings on patient empowerment and trust dynamics in SDM e) Discussion of how symptom severity might influence preference stability\nLimitations of My Review: I explicitly acknowledge that I cannot evaluate:\nThe mathematical validity of the models presented The statistical methodology The theoretical framework of utility values; these aspects require separate review by appropriate experts.\nConclusion: While the clinical implications of this work are important and well-supported by recent research on patient empowerment and SDM, I can only partially approve this paper, specifically regarding its clinical relevance and practical implications. The mathematical components require separate expert review and validation. The integration of recent findings on patient empowerment and trust dynamics in SDM strengthens the paper's clinical relevance while highlighting the complexity of patient preference elicitation in clinical settings.\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 source data required\n\nAre the conclusions drawn adequately supported by the results? Partly",
"responses": []
}
] | 1
|
https://f1000research.com/articles/11-1533
|
https://f1000research.com/articles/11-1531/v1
|
19 Dec 22
|
{
"type": "Systematic Review",
"title": "Elder abuse in Saudi Arabia: A systematic review",
"authors": [
"Ali S. Alqahtani",
"Ammer L. Alanazi",
"Ziyad K. alsayyali",
"Khalid Z. Almubarak",
"Nasser N. Alzahrani",
"Amr R. Bokhari",
"Ritesh G. Menezes",
"Ammer L. Alanazi",
"Ziyad K. alsayyali",
"Khalid Z. Almubarak",
"Nasser N. Alzahrani",
"Amr R. Bokhari",
"Ritesh G. Menezes"
],
"abstract": "Background: Elder abuse is a common global problem that is certainly preventable. The first step to tackling elder abuse is by researching its prevalence and acknowledging it as a prevailing problem. However, there is a lack of studies on elder abuse in Saudi Arabia. In this review, the socio-forensic problem of elder abuse in Saudi Arabia is addressed. Methods: The PubMed database was systematically searched for articles on elder abuse in Saudi Arabia from inception to 1 July 2022. Search terms included “elder abuse”, “elderly abuse”, “geriatric abuse”, “aged abuse”, “senior abuse”, and “elder maltreatment”. In addition, to detect studies reported from Saudi Arabia, the following search terms were included: “Saudi Arabia”, “KSA”, and “Kingdom of Saudi Arabia”. The search results were screened for relevant articles. Studies from Saudi Arabia that addressed elder abuse were included in this review. Studies from other countries and studies that addressed abuse in other age groups were excluded. Results: A very high percentage of the elderly in shelter homes were found to have experienced abuse, especially psychological, which resulted in an array of mental health implications such as anxiety, depression, and sleep disorders. A high level of awareness of elder abuse, in addition to a high sense of responsibility towards the elderly, was reported. Conclusions: As the elderly represent a significant percentage of the population in Saudi Arabia, and with the community being heavily family-centered, it is recommended to increase dedicated efforts towards nationwide research on elder abuse and to formulate effective national programs to ensure its prevention in Saudi Arabia.",
"keywords": [
"older adults",
"abuse of older people",
"elder mistreatment",
"geriatrics",
"abuse",
"neglect",
"Saudi Arabia",
"systematic review"
],
"content": "Introduction\n\nElder abuse is generally defined as “any direct action, inaction, or neglect against the elderly that causes harm or puts them in danger of harm, either by a person in a position of presumed trust or by any other person who targets the elderly because of their age or disability”.1 Older adults are at a high risk of being abused, which is a serious, but preventable socio-forensic problem. Elder abuse, like other forms of domestic or interpersonal violence, is a global phenomenon that crosses different cultural and socioeconomic backgrounds.2 Elder abuse is categorized into various types, mainly, psychological, physical, sexual, financial, and neglect, depending on the act by the abuser.3 A systematic review and meta-analysis that included 52 studies from 28 countries across the globe reported an estimated prevalence rate of 15.7% for overall elder abuse in the community setting.4 The prevalence estimates for psychological abuse, physical abuse, sexual abuse, financial abuse, and neglect were 11.6%, 2.6%, 0.9%, 6.8%, and 4.2%, respectively, in the elderly in the community setting.4 According to another global systematic review and meta-analysis, the prevalence estimates for psychological abuse, physical abuse, sexual abuse, financial abuse, and neglect were 33.4%, 14.1%, 1.9%, 13.8%, and 11.6%, respectively, in the elderly in the institutional setting.5\n\nThe objective of this systematic review was to summarize the socio-forensic problem of elder abuse in Saudi Arabia based on a literature survey.\n\n\nMethods\n\nWe followed the checklist of Preferred Reporting Items for Systematic Review and Meta-Analysis Protocols (PRISMA-P) (Supplementary file 1) in designing this systematic review (https://prisma-statement.org/). This systematic review was not pre-registered in the International Prospective Register of Systematic Reviews (PROSPERO).\n\nThe PubMed database was systematically searched for articles on elder abuse in Saudi Arabia from inception to 1 July 2022 (the date of literature search). The search terms included “elder abuse”, “elderly abuse”, “geriatric abuse”, “aged abuse”, “senior abuse”, and “elder maltreatment”. In addition, to detect studies reported from Saudi Arabia, the following search terms were included: “Saudi Arabia”, “KSA”, and “Kingdom of Saudi Arabia” (Table 1). The search results were screened for relevant articles. The steps followed in the PubMed search for relevant literature are depicted in Figure 1. Other databases like Scopus and Web of Science, registers, websites, organizations, and reference lists were not searched to identify additional studies.\n\nWe included cross-sectional and observational studies that addressed elder abuse in Saudi Arabia. Studies conducted in countries other than Saudi Arabia and those that addressed abuse in other age groups were excluded. Case reports, editorials, letters, and review articles were also excluded. There were no filters based on the publication date and language applied. Three reviewers (ASA, ALA, ZKA) independently screened the title and abstract to decide whether the articles would be included in our review.\n\nIn a preliminary search of PubMed, 116 records were identified. The irrelevant articles were removed (n=108). Three records were excluded as they were conducted in other countries (n=3). Two records were excluded as they included another age group (n=2). The PRISMA flow chart (Figure 1) outlines the screening process. Three reviewers (ASA, ALA, ZKA) independently screened the title and abstract of the records identified in the first phase and subsequently screened the full text of the potentially eligible studies; disagreement, if any, was resolved in consultation with the other reviewers (KZA, NNZ, ARB, RGM). The records were screened manually, and no automation tool was used in this process. The authors or study investigators were not consulted to confirm the data or to obtain additional data.\n\n\nResults and discussion\n\nThe search string “(elder abuse OR aged abuse OR elderly abuse OR senior abuse OR elder maltreatment OR geriatric abuse) AND (Saudi Arabia OR KSA OR Kingdom of Saudi Arabia)” identified 116 records at PubMed, of which 3 articles6–8 met the inclusion criteria (Table 2).\n\nAlraddadi conducted a cross-sectional study that included 446 elderly participants from 43 sheltered homes in Makkah and Jeddah.6 The study found that 81% of the participants had experienced at least one type of abuse in the last 12 months, and the most common type of abuse was psychological abuse (71%) and the least was sexual abuse (0.01%). In addition, other types of abuse were recorded such as neglect (67%), financial abuse (54%), and physical abuse (13%).6 The common risk factors associated with elder abuse were being female and having a chronic disease.6 Another report7 by Alraddadi on the same number of participants at the same sites6 provided details on the assessment of the impact of abuse on psychological and physical health among older adults living in the institutional setting.7 The study found that 83% of older adults who suffered from any type of abuse had symptoms of depression, 62% had anxiety disorders, and 59% had problems with sleep.7 However, only 4% of older adults who suffered from any type of abuse had suicidal ideation in the last 12 months.7 The study also found that 46% of older adults who suffered from any type of abuse reported poor physical health and 48% reported increased utilization of healthcare.7 Poor physical health was significantly associated with psychological abuse, physical abuse, financial abuse, and neglect. However, increased utilization of healthcare was not significantly associated with psychological abuse.7 Almakki et al. conducted a cross-sectional study on 430 participants at various primary healthcare centers in Qatif in the Eastern Province of Saudi Arabia.8 The aim of the study was to identify the knowledge of elder abuse and attitudes towards it among the adult attendees of the aforementioned primary healthcare centers. The study found that nearly all the participants considered it to be their responsibility to report witnessed elder abuse and over 90% of the participants considered elder abuse to be a criminal act.8\n\nMany studies have investigated the prevalence of elderly abuse in multiple countries. For instance, a study conducted in Nepal reported a 54.5% prevalence of overall elder abuse.9 Neglect (23.1%), psychological abuse (20.6%), physical abuse (6.5%), financial abuse (2.4%), and sexual abuse (1.9%), were the types of abuse among the elderly.9 In Saudi Arabia, these figures were much higher, except for sexual abuse.6 Physical abuse and psychological abuse were more common among elderly females in Nepal.9 A study in Egypt reported that 43.7% of the elderly were abused.10 The most common form of elder abuse was neglect (42%), followed by physical abuse (6%), psychological abuse (5%), and financial abuse (4%), with females reporting greater abuse than males.10 Similarly, the female gender was found to be a common risk factor for elder abuse in Saudi Arabia.6 A systematic review and meta-analysis on the prevalence of elder abuse in Iran reported an estimated 48.3% prevalence of overall elder abuse11 which is much less than the 81% reported in the single study conducted in Saudi Arabia.6\n\nWe acknowledge the limitation of including only a single database in the review process. Other prominent databases such as the Scopus and Web of Science were not included in the search strategy. The use of self-report questionnaires or interviews adopted in the methodology related to data collection in the three studies that were included in the present review was a major limitation in terms of the evidence.\n\n\nConclusions and recommendations\n\nElder abuse is a topic that is understudied in Saudi Arabia. Nevertheless, limited studies did prove the occurrence and severity of elder abuse in the institutional setting in the country. Elder abuse was common in sheltered homes. In the institutional setting, the female gender and those suffering from chronic illnesses were the two most common elderly cohorts vulnerable to abuse. Anxiety, depression, and sleep disorders were common in those abused older adults in the institutional setting in Saudi Arabia.\n\nThere is a lack of research on the prevalence and risk factors of elder abuse in the community setting in Saudi Arabia. More studies, particularly nationwide, are required to more accurately document the prevalence, risk factors, types, characteristics of elder abuse, and its impact on the psychological and physical well-being of the elderly population in both institutional and community settings in the country. Research should also be conducted to study anatomic locations of physical elder abuse. It is important to recognize the burden of elder abuse to formulate adequate and effective strategies to ameliorate elderly care in Saudi Arabia.",
"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: Elder abuse in Saudi Arabia: A systematic review, https://doi.org/10.6084/m9.figshare.21621306.v5. 12\n\nThis project contains the following extended data:\n\n- PRISMA flowchart\n\nFigshare: Elder abuse in Saudi Arabia: A systematic review, https://doi.org/10.6084/m9.figshare.21621306.v5. 12\n\n- PRISMA Checklist Elder Abuse.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\nJohnson MJ, Fertel H: Elder abuse. StatPearls. Treasure Island, FL:StatPearls Publishing; 2022. (accessed on 15 October 2022).\n\nNational Research Council: Elder Mistreatment: Abuse, Neglect, and Exploitation in an Aging America. Washington, DC:The National Academies Press;2003. (accessed on 15 October 2022). Publisher Full Text\n\nBlundell A, Gordon A: Geriatric Medicine at a Glance. Hoboken, NJ:Wiley-Blackwell;2015.\n\nYon Y, Mikton CR, Gassoumis ZD, et al.: Elder abuse prevalence in community settings: a systematic review and meta-analysis. Lancet Glob. Health. 2017; 5: e147–e156. PubMed Abstract | Publisher Full Text\n\nYon Y, Ramiro-Gonzalez M, Mikton CR, et al.: The prevalence of elder abuse in institutional settings: a systematic review and meta-analysis. Eur. J. Pub. Health. 2019; 29: 58–67. PubMed Abstract | Publisher Full Text | Free Full Text\n\nAlraddadi K: Prevalence and risk factors of elder mistreatment in sheltered homes. J. Interpers. Violence. 2022; 37: 1588–1603. PubMed Abstract | Publisher Full Text\n\nAlraddadi K: Impacts of mistreatment on the psychological and physical health of older adults living in sheltered homes. Geriatr. Nurs. 2022; 43: 182–187. PubMed Abstract | Publisher Full Text\n\nAlmakki ZE, Alshehri SZ, Abdel Wahab MM: Knowledge and attitudes regarding elder abuse in the community, Eastern Province Saudi Arabia. BMC Geriatr. 2020; 20: 85. PubMed Abstract | Publisher Full Text | Free Full Text\n\nAcharya SR, Suman BK, Pahari S, et al.: Prevalence of abuse among the elderly population of Syangja, Nepal. BMC Public Health. 2021; 21: 1348. PubMed Abstract | Publisher Full Text | Free Full Text\n\nAbdel Rahman TT, El Gaafary MM: Elder mistreatment in a rural area in Egypt. Geriatr. Gerontol. Int. 2012; 12: 532–537. PubMed Abstract | Publisher Full Text\n\nAbdi A, Tarjoman A, Borji M: Prevalence of elder abuse in Iran: a systematic review and meta-analysis. Asian J. Psychiatr. 2019; 39: 120–127. PubMed Abstract | Publisher Full Text\n\nAlqahtani AS, Alanazi AL, Alsayyali ZK, et al.: Elder abuse in Saudi Arabia: A systematic review. figshare. Online resource.2022. Publisher Full Text"
}
|
[
{
"id": "196436",
"date": "01 Sep 2023",
"name": "Fuad Abujarad",
"expertise": [
"Reviewer Expertise Elder Abuse"
],
"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 used comprehensive study search terms and accurate method for study selection however they only focused on one single database in the search process. That would have been fine if the volume of paper that will end up being included in the analysis is sufficient however they ended with including three papers only (two from one author). This low number of identified and analyzed papers is not sufficient to draw a comprehensive and dependable scientific conclusion for a systematic review. The authors recognize this limitation, and they say that they only use a single database in the review process. My recommendation is that this article will be presented as a scoping review, since in reality there may not be other studies about elder abuse in that region.\nThe other option is to include more databases and other per reviewed publications from local and regional scientific conference proceedings to try to identify more studies about elder abuse in Saudi Arabia.\n\nAre the rationale for, and objectives of, the Systematic Review clearly stated? Yes\n\nAre sufficient details of the methods and analysis provided to allow replication by others? Yes\n\nIs the statistical analysis and its interpretation appropriate? Partly\n\nAre the conclusions drawn adequately supported by the results presented in the review? Partly",
"responses": []
},
{
"id": "208244",
"date": "25 Oct 2023",
"name": "Sonia Salari",
"expertise": [
"Reviewer Expertise Expertise in elder abuse",
"neglect and exploitation. Family violence across the life course. Gerontology",
"including articles about Middle Eastern immigrants and Arab Americans."
],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address 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 a meta-analysis of the mention of elder abuse in Saudi Arabia. I think the argument would be better accompanied by an introduction literature review to set up the Saudi culture for readers who may be unfamiliar. What aspects of that society might be related to the experience of abuse, neglect or exploitation among those in the elder adult generation?\n\nThe limitation of using just one database PubMed was not explained. Rather than explain it, why not use them to search for articles from Saudi Arabia and see if any others can be discovered?\n\nA major message for this research would be to encourage further research to study elder mistreatment in this target Arab country. We need a proper lead up to why we should be concerned, or what protective factors might exist. We should be aware that societal prohibitions related to sexual expression in general, might lead elders to under report sexual assaults, etc. Perhaps oppression based on gender might lead to isolation of elders, particularly the women. Women may be the designated caregivers for sick elders. What happens if a family does not have daughters-in law or female offspring? But by far, the biggest issue is the paucity of research about later life experiences in this country.\n\nAre the rationale for, and objectives of, the Systematic Review clearly stated? Partly\n\nAre sufficient details of the methods and analysis provided to allow replication by others? Yes\n\nIs the statistical analysis and its interpretation appropriate? Not applicable\n\nAre the conclusions drawn adequately supported by the results presented in the review? Partly",
"responses": []
},
{
"id": "208249",
"date": "14 Sep 2024",
"name": "Minna-Liisa Luoma",
"expertise": [
"Reviewer Expertise Elder Abuse",
"Violece and abuse against people with disability",
"quality of life",
"elderly care"
],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe paper is well written, proceeds logically and easy to read. Topic is important in all over the world and difficult to research. I find it good that paper concentrates only SA, since it could have more impact and raise awareness in Saudi Arabia. How ever the limitation is that there is only very few studies on this issue and the researches have acknowledged it and using only one database. Conclusions are sound. I hope research on the prevalence and risk factors of elder abuse in the community setting could be conducted in SA in the near future. In Introduction I would use this WHO:s definition : The abuse of older people, also known as elder abuse, is a single or repeated act, or lack of appropriate action, occurring within any relationship where there is an expectation of trust, which causes harm or distress to an older person. This type of violence constitutes a violation of human rights and includes physical, sexual, psychological and emotional abuse; financial and material abuse; abandonment; neglect; and serious loss of dignity and respect..\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": []
}
] | 1
|
https://f1000research.com/articles/11-1531
|
https://f1000research.com/articles/11-1528/v1
|
19 Dec 22
|
{
"type": "Research Article",
"title": "The family situation of wards under the supervision of court - appointed custodians during COVID-19 pandemic in Poland: a self-report study to examine the perception of probation officers.",
"authors": [
"Robert Opora"
],
"abstract": "Background: The Polish Probation Service is strongly linked to social work. This text presents how the work and the problems of clients have changed in the Polish Probation Service during the COVID-19 pandemic. The article presents empirical data. Methods: A questionnaire was used among 216 Polish probation officers. Results: The obtained results describe the changes in the problems experienced by clients of the Probation Service during the COVID-19 pandemic. Conclusions: Since we can expect further increase in the frequency of occurring personal and social problems experienced by court-appointed custodians’ wards, financial resources and energy of social institutions, cooperation with court guardianship should be focused on psycho-educational campaigns concentrating on providing information on symptoms of problems in the mental and emotional sphere. Preventing exclusion of groups at risk in the labour market and supporting families to prevent the escalation of psychopathology, development of addictions and domestic violence should be priority areas.",
"keywords": [
"Pandemic COVID – 19",
"Social problems",
"Probation supervision",
"family",
"social rehabilitation"
],
"content": "Introduction\n\nAlthough we do not yet know all of the consequences of the COVID-19 pandemic, after over a year we can begin to describe the reality we find ourselves in. The COVID-19 pandemic was a global issue. Related experiences affected every person irrespective of their country and community and forced us to change our lifestyles and ways of working. People have faced pressures in restricting social interactions, especially with their relatives, due to the necessity of limiting virus transmission.\n\nThe Polish Probation Service is strongly linked to social work. Similarly to all social and professional groups, during the pandemic the Probation Service had to adjust to limitations concerning public health. This entailed limitations on physical contact and increased dependency on remote supervision.\n\nSimultaneously, wards, especially those with the most complex needs, experienced a decrease in emotional wellbeing which worsened as a result of the isolation and limited access to institutional forms of support (Carr, 2021). The area of interpersonal relations which usually supports people in difficult situations, was limited. It is of a particular significance especially for persons who do not easily establish social relations, and for whom workplace or school and participation in out-of-school activities provided space in which these relations can be established.\n\nSocial distancing has made it difficult to maintain relationships and a growing feeling of loneliness. Pandemic-related fear and uncertainty has a negative impact on the family system. People may fear for the health and life of their grandparents or parents (Ptaszek et al., 2020). Furthermore, the restrictions and related isolation may reinforce or disclose family dysfunctions.\n\nIn many European countries, probation is an important element of the social work system. Hence, the concept of social work as work with people who violate social norms is common (Vanstone, 2012). The tasks of probation officers include enabling their clients who violate social norms to overcome difficult life situations using their own resources and abilities. The probation officers perform their work with families experiencing various social issues such as bad economic situations, unemployment, addiction, breakdown of social and family relations or violation of legal orders (Janus-Dębska and Gronkiewicz-Ostaszewska, 2019). The ward’s family situation is influenced by the quality and quantity of activities undertaken by a court-appointed custodian. Limitations resulting from the restrictions introduced during the pandemic affected various spheres of human functioning.\n\nDue to social distancing, closure of workplaces, remote work and introduction of distanced teaching, families faced new challenges. In case of some families or particular members thereof these circumstances contributed to the occurrence of a crisis or reinforced existing ones. In response to these challenges, the institution of probation has adapted, struggled, and ultimately endured to continue serving the court and the community.\n\nDescribing the reality of each individual area of Poland which experienced the pandemic is not possible because of the limited research. This study aims to capture generally what practice looked like pre-pandemic and how the discipline responded when confronted by this novel challenge. As terms like “social distancing,” and “quarantine” were added to the country’s lexicon, the probation officers raced to keep the courts running, rights preserved, and communities safe. Some of the responsibilities of the probation officers were possible to run via video conferences. However, as anyone who has used video conferencing software is aware, virtual communication is rarely a substitute for in-person interaction. While some may determine that the benefits of not having to travel outweigh the costs of having an online meeting, we do not, and cannot, find a balance with client rights and support (Whaley et al., 2021). Assessment interviews are now completed by videoconferencing or over the phone. Neither of these options is ideal; quickly building rapport with a client is much more difficult in a virtual environment. However, videoconferencing does offer some parallels to being in person. Being able to see one another allows both parties to give and receive non-verbal feedback such as nodding or leaning in, it assists in distinguishing between whether the speaker is taking a thoughtful pause or has finished speaking. Although videoconferencing is significantly different from in-person communication, a probation officer can create a trusting and comfortable environment for the defendant over video. Unfortunately, doing so becomes difficult when interviews are conducted over the phone. There is no longer body language or non-verbal feedback; accidental interruptions become more frequent; and a sense of connection is even more difficult to foster. The pandemic posed significant complications in the way officers carried out their duties.\n\nAs we see in Figures 1 and 2, both for adult and juvenile probation officers in 2022, compared to 2019, the number of new cases reported and completed cases decreased. Whether the decrease in adjudications was a direct result of reduced procedural efforts (stemming from a shift in investigation and charging practices by the executive branch and other agencies) or limitations on in-person court proceedings related to the pandemic is unclear.\n\nSource: Own analyzes based on the statistics of the Ministry of Justice, Department of Strategy and European Funds, MS-S40r Report on the activity of the probation court service for the year 2019, 2020. Warszawa.\n\nSource: Own analyzes based on the statistics of the Ministry of Justice, Department of Strategy and European Funds, MS-S40r Report on the activity of the probation court service for the year 2019, 2020. Warszawa.\n\nThe impact of a reduced caseload and the constraints posed by the pandemic for many probation officers did not mean a decrease in work. The pandemic made their work more complex because access to most social services became limited and probation officers had to look for alternative interventions for their clients. Unsurprisingly, officers used the temporary decrease in caseload to take on additional duties, and to assist their clients more because a lot of other social agencies closed themselves at the beginning of the pandemic.\n\nThe aim of the research was to learn opinions of probation officers regarding changes in the scope of social problems encountered by them in their performance of tasks during the pandemic. Thus, the following research question was asked:\n\nHave social problems in families under the supervision of a court-appointed custodian intensified during the pandemic?\n\n\nMethods\n\nThe research was conducted in 12 randomly selected court regions in Poland among 216 professional court-appointed custodians. The questionnaire was sent to all probation officers from these court regions. A total of 482 probation officers received the questionnaire, out of which 216 returned them. All of them were completed because the system didn’t allow uncompleted questionnaires to be returned.\n\nThe study was of a quantitative nature and was conducted using a diagnostic anonymous survey developed for the purposes of the research. The link with a survey questionnaire was developed in Microsoft Forms and shared by the University of Gdańsk with the chosen offices of District Probation Officers in an electronic form. Then the questionnaires were sent by e-mail by the office of District Probation Officers to all probation officers from the district. The probation officers were asked to fill in the questionnaire by using the link attached to the email with the instructions. The research began on August 01, 2021 and lasted one month until August 30, 2021. The research was anonymous and voluntary. A copy of the questionnaire can be found under Extended data (Opora, 2022).\n\nThe research was conducted in 12 randomly selected court regions in Poland among 216 of 480 professional court-appointed custodians. To be eligible for the study, respondents had to be a professional probation officer. District courts participating in the study were randomly selected from 46 districts in Poland. A potential source of bias in the research was the respondents’ limited work experience. They had no more than seven years of work experience in the probation system.\n\nOn 13 July 2021, the study received the approval of the Ethics Committee of the Faculty of Psychology of the Gdansk University, Poland (approval no. 20/2021). Participants gave written informed consent for the use and publication of their data.\n\nThe research was conducted in August 2021, that is, after approximately 1.5 years since the announcement of the pandemic in Poland. The survey was created and completed using the MS Forms platform. The link was sent out through e-mail by the secretariats of the district courts to all family probations officers from the chosen districts.\n\nBefore the final research, a pilot study was conducted among 12 custodians representing both criminal as well as family and juvenile teams. As a result of the obtained information and undertaken activities, the survey questionnaire was partially modified and adjusted. In the metric of the questionnaire, an additional question about occupational rank was introduced and a question about drug abuse was added. The final version of the questionnaire included a metric on basic demographic information such as gender and length of employment as a family probation officer and the main part of the questionnaire consisted of 16 questions to measure the variable defined as the family situation of probationers’ wards. Respondents were allowed to reply to each question by selecting one answer from four. The answers were as follows: “it was not before and it is not now”, “it was less before than now”, “it was the same before as now”, “it was more before than now”. In addition, the text was stylistically corrected.\n\nAt the beginning of the survey, questionnaire instructions informed respondents of the purposes and rules of conducting research. Furthermore, respondents were informed of the email address of the person responsible for the research implementation. At the beginning of the survey questionnaire, a request for consent to participate in the research was included. The respondent could at any time stop his or her participation by closing the website and not sending the results.\n\nThe statistical analysis was conducted through Statistica software. Basic descriptive statistics were primarily used and, in order to determine statistical significance of differences in obtained frequency of answers to questions, chi2 test was used. The reliability of the final version of the questionnaire was tested using Cronbach’s Alpha 0.89 which indicated high internal consistency. The number of potentially eligible probation officers in the 12 selected districts was 480. The number of confirmed eligible probation officers was 480, and the number included in the study who completed questionnaires was 216.\n\nSince the research was conducted online, only completed questionnaires could be submitted. Therefore, there was no problem regarding lack of data or incomplete data in collected answers. There was just one summary measure over time.\n\n\nResults\n\nIn the research undertaken, the majority of the respondents were women (70%), while men made up 30%.\n\nIn order to determine problems experienced by wards during the pandemic, custodians gave answers by comparing their current work experiences to experiences from before the pandemic. The full dataset can be found under Underlying Data (Opora, 2022).\n\nAs shown in the Table 1, 55% of custodians participating in the research, the number of difficulties related to wards staying with demoralised persons during the pandemic has not changed since before the pandemic. However, according to 40.27% of custodians, these difficulties intensified during the pandemic. Some persons, especially students, at the time of introducing remote education were probably at a disposal of significantly more time and remained outside of the control of a school or parents. Among the respondents, 2.77% custodians stated that currently they have more difficulties with regard to their wards staying with demoralised persons. The test of statistical significance indicates an existing difference, which means that during the pandemic, despite recommendations regarding social distance, a number of problematic behaviours resulting from leaving juveniles with demoralised peers increased (chi2=96.93; df=2; p<0.05).\n\nEvery second respondent (54.62%) stated that as a result of the pandemic they observed the deteriorating financial situation of families under supervision/custody. Whereas 2.77% respondents indicated that during the pandemic the situation of families under their care improved. Other respondents stated that the financial situation of families under supervision/custody from before the pandemic and during the pandemic has not changed. The distribution of this frequency indicates statistically significant differences (chi2= 95.16; df=2; p<0.05) and allows us to draw conclusions on the deteriorating financial situation of custodians’ wards.\n\nIn the opinion of 67.12% of custodians participating in the research, the number of conflicts in families increased. Only 0.45% noticed a drop in this area during the pandemic. Other respondents did not indicate any change before pandemic and during the pandemic in this scope. The changes indicate differences in the distribution of given answers (chi2=146.37; df=2; p<0.05), which proves the increase in conflicts among families under supervision or custody.\n\nIn the opinions of 45.83% of court-appointed custodians, alcohol abuse by wards’ family members increased. Only 0.46% of them claim that the abuse decreased and 52.31% believe that this problem remains at the same level. These results indicate the presence of an alcohol problem in families with regard to whom a court-appointed custodian was adjudicated. This problem has been additionally growing under the circumstances resulting from the limitations introduced due to the pandemic. The test of statistical significance indicates a significant difference in the scope of given answers (chi2=104.90; df=2; p<0.05).\n\nOn the grounds of obtained results, it can also be concluded that the problem of alcohol abuse by wards increased. Such an opinion was given by 44.44% of court-appointed custodians. According to only 1.38% of respondents, this problem has decreased during the pandemic. Whereas 53.24% of respondents believe that this problem has already existed before the pandemic and continues to exist at the same level. Changes in the distribution of given answers are statistically significant (chi2=100.72; df=2; p<0.05) and indicate the growing problem of alcohol abuse during the pandemic by persons with regard to whom a supervision of a court-appointed custodian has been adjudicated.\n\n35.64% of court-appointed custodians are of the opinion that the use of narcotics and designer drugs has also increased. Only 2.77% believe that the situation improved during the pandemic and 58.33% state that they encounter this problem during the pandemic as often as before the pandemic. Differences in the distribution of given answers are statistically significant chi2=104.51; df=2; p<0.05, which indicates that, in the court-appointed custodians’ opinion, the pandemic period intensified the problems of using narcotics and designer drugs.\n\nAs many as 74.53% of court-appointed custodians believe that the problem with not participating in school classes or inactivity during classes, increased. Only 1.85% of custodians believe that this problem decreased during the pandemic and 13.42% stated that this problem remained at the same level. These results indicate significant differences in the distribution of given answers (chi2=220.09; df=2; p<0.05).\n\n55.55% of court-appointed custodians observed while performing supervisions and custodies a growing professional passivity in families, 38.88% of custodians notice existence of this problem and believe that it currently remains at the same level as before the pandemic. Only 1.85% of respondents believe that this problem decreased during the pandemic. Thus undoubtedly, during the pandemic court-appointed custodians have observed a growing professional passiveness in families under supervisions or custodies (chi2=101.69; df=2; p<0.05).\n\nDuring the pandemic every third respondent (37.5%) has observed a growing frequency of reporting complaints by the family to the custodian due to the improper behaviour of the ward and 56.48% have noticed that this problem is at the same level during the pandemic as it was in the period before the pandemic. Only 2.31% of custodians responded that the number of these complaints is currently smaller. The obtained results indicate the growing number of problems during the pandemic engaging their attention and time. Differences in the distribution are statistically significant (chi2=101.66; df=2; p<0.05).\n\n26.85% of respondents perceive the period of the pandemic as the one in which referring to educational-care and resocialization facilities or revoking custody due to the non-fulfilment of obligations imposed on the ward are more frequent. 4.62% of studied custodians do not notice this trend and believe that the number of such interventions dropped. While 59.72% of respondents believe that interventions are as frequent as before the pandemic. Differences in the obtained distribution are statistically significant (chi2=109.17; df=2; p<0.05).\n\nAs Figure 3 ilustrates, during the pandemic court-appointed custodians have noticed a significant growth in domestic violence (chi2=104.56; df=2; p<0.05). Every second respondent (56.94%) agrees that they have more often encountered this phenomenon during the pandemic than before the pandemic. Only 1.85% believe that there are fewer violent behaviours in a family during the pandemic, while 39.35% state that this problem is currently present to the same extent as before the pandemic.\n\nFurthermore, the number of aggressive behaviours of wards towards household members increased. It is stated by every second respondent (49.07%), and 46.75% of studied custodians assess the occurrence of this problem as the same as before the pandemic. Only 1.85% of custodians believe that this problem occurred more frequently before the pandemic. The obtained distribution of the frequency of answers is significantly different statistically (chi2=94.02; df=2; p<0.05). On the one hand, pandemic conditions forced bigger social distance in the public space, but at the same time resulted in family members spending more time at home in small spaces, which forced more interactions that have not always been comfortable.\n\nThe increase in aggressive behaviours in wards during the pandemic occurred not only towards the closest family members. Every third custodian (33.33%) observed an increase in aggressive behaviours of wards towards other persons they know and 56.01% assess this problem as remaining at the same level. Only 2.31% of respondents believe that the number of such behaviours dropped during the pandemic. Differences in the obtained distribution are statistically significant (chi2=102.76; df=2; p<0.05).\n\nThe scale of the hate phenomenon cyberbullying has also increased during the pandemic. This means that clients of probation officers more often used aggressive and negative comments on the internet. It was observed by 41.66% of respondents, whereas 43.98% believe that this problem occurs as frequently as before the pandemic and does not differ from before the pandemic. Only 1.85% of persons stated that this phenomenon currently occurs less frequently than before the pandemic. Obtained results indicate the existence of significant differences in the distribution of the frequency of answers (chi2=83.08; df=2; p<0.05).\n\nAdditionally, custodians’ answers imply that 40.27% believe that their wards spend free time in a risky manner more frequently than in the period before the pandemic and 49.07% believe that their wards spent time in a risky manner before the pandemic and continue to do so. Only 5.09% of respondents believe that wards currently practice social distancing during their free time than before the pandemic. The distribution of obtained answers indicates the presence of statistically significant differences (chi2= 74.32; df=2; p<0.05).\n\nAs many as 69.44% of court-appointed custodians observed an increase in the mental overload experienced by the loved ones of their wards. Perhaps this overload was caused by external restrictions resulting from the introduction of the pandemic, as well as growing interpersonal, family or personal problems resulting from the complications of restrictions. According to 25.04% of custodians the mental overload of their wards occurred before the pandemic and currently occurs with the same frequency. While 2.31% of custodians believe that the pandemic is favourable for the families of their wards and mental overload in these families occurred more frequently before the pandemic than currently. The obtained distribution of the frequency of answers indicates statistically significant differences (chi2=156.18; df=2; p<0.05).\n\nIn summary, it can be seen that during the family situation during the pandemic, according to custodians participating in the research, problems with wards’ participation in school were growing the most (74.53%). The next position was taken by the mental overload of the ward’s loved ones (69.44%). Family conflict (67.12%) and domestic violence (56.94%) were ranked lower. Another place was taken by professional passiveness (55.55%) and worsening financial situation of families (54.62%). Another group of difficulties encountered by wards’ families relates to aggressive and risky behaviours, as well as alcohol abuse. These behaviours have increased in the opinion of approximately 40% of respondents. According to 37.5% of studied custodians, during the pandemic the frequency of reporting complaints by the family to the custodian with regard to the improper behaviour of the ward increased.\n\nOne-quarter of respondents (26.85%) believe that, during the pandemic, there was an increase in the incidence of wards being referred to residential educational care and rehabilitation facilities.\n\n\nDiscussion\n\nThese results show that the COVID-19 pandemic resulted in a lot of changes among families under the supervision of a court-appointed custodian and a number of difficulties faced by the custodians occurred. Social problems that were disclosed during the COVID-19 pandemic are related to the prolonged exposure to worrying information, great uncertainty and life and health threatening conditions which cause various types of losses (Hamer and Baran, 2021). Almost eight in ten adults identified the coronavirus pandemic as a significant source of stress in their lives (Gruber et al., 2021). Epidemic and related sanitary requirements cause problems in various spheres of human life: social, economic, cultural, as well as the education of children, youth and adults.\n\nDuring the pandemic, on the grounds of the conducted research, an increase in the consumption of alcohol and intoxicants by court-appointed custodians’ wards and their family members has increased. This was a way to ease the tension easily and quickly (Al’Absi, 2010).\n\nFurthermore, we observe an escalation in the number of family conflicts, aggressive behaviours and domestic violence due to the lack of ability to solve conflicts through dialogue and negotiations. It may be assumed that the escalation of the violence is additionally affected by: isolation of persons in small housing facilities, drug and alcohol abuse, a deteriorating financial situation, intensification of problems that were present before the pandemic, lingering fear and living in stress, and untreated mental disorders. As indicated by the results of the presented research, during the pandemic the custodians’ wards usually remained under the influence of other demoralised persons, experienced professional inactivity, avoided participating in school classes and thus, have had more time and have remained outside of the social control. Such circumstances favour demoralisation and the occurrence of aggressive behaviours (Urban, 2000).\n\nResults of the survey indicate the growing phenomenon of cyberbullying which is increased by remote education, social distance and remote work (Ahmed and Braithwaite, 2004). As a result of a lack of direct contact with other persons, interpersonal communication is transferred online and is reflected in social media. In the case of mediated communication, there is less sense of a partner’s presence in the communication (Pyżalski, 2012). This leads to less focus on the person who we communicate with and mutual relations with this person. Additionally, perpetrators of online aggression usually feel anonymous which favours the occurrence of the phenomenon of disinhibition (Aronson, 2005).\n\nMoreover, we observe a decrease in motivation among custodians’ wards and their families to participate in educational classes and gainful employment. In the educational area the initial fascination with remote education resulting from the relatively easy way to obtain positive grades, may be reduced in ambitious children. Whereas children with specific needs have been to a large extent deprived of rehabilitation classes, skills training or early development support. Due to various reasons, many parents are not able to help their children with assignments, which is why they are left alone in this area as well.\n\nThe constantly changing situation makes it easier for children and youth to assume the following: “what is the point in trying and learning, if no one knows what will happen next”. They experience promises of return to schools and then, it turns out it is not possible. They notice that adults do not know what will happen. In effect, it may be assumed that some students log in remote classes and have no motivation to actively participate in them or do not log in at all.\n\nUsually, every difficult situation brings some benefits. Online school education is comfortable for students characterised with a deficit in motivation. They do not have to commute to school. Consequently, these students may find it difficult to return to school, since they have developed strategies of functioning outside of the school. They can avoid stressful or uncomfortable situations which they encountered in school by using technical difficulties as excuses.\n\nWhereas, as a result of stopping many interrelated industries, a lot of people lost their jobs or experienced a forced break in professional activity, which significantly affected the economic situation of many families (GUS, 2020: 1). In a short time, the employee market, in which we observed many job offers, changed into the employer market (GUS, 2020: 1). Thus, currently an employee has to make an effort to be employed and an employer does not strive for employment. In such a situation, court-appointed custodians’ wards, who are usually characterised with deficits in professional and personal competences, belong to the group who will be, among others, affected by the consequences of this change. The longer the pandemic lasts, the bigger economic diversification we can expect, where persons with high qualifications, intellectual and adaptive potential, who are technologically adept will adjust easier to new challenges.\n\nRemaining in the conditions of the pandemic and staying with family members in small spaces deteriorated the functioning not only of custodians’ wards but also their families. Personal, family and interpersonal problems of wards under supervision of court-appointed custodians, which have been growing during the pandemic, lead to the intensified mental overload of their family members. Despite the implementation of a series of procedures and instructions for the period of the pandemic by the court guardianship, we cannot forget that each form of social isolation can lead to the occurrence of emotional, social and cognitive disorders (Pratt, 2009). At the same time, the pandemic limited access to health care and psychotherapy for persons in these risk groups. Thus, in some cases support provided by the court-appointed custodian could have been the only available form of help.\n\nOne strength of the current study was its large sample, which included probation officers from all over Poland, specializing both in adult as well as in family and juvenile cases. This allowed for capturing the specifics of supervising clients of probation officers during the pandemic. However, the current study also has some limitations. One is the fact that it involved only probation officers in Poland. This has already been addressed in an ongoing research project seeking to include probation officers from other countries. Another limitation comes from the method of collecting data via the internet. The link with the questionnaire was sent to the business e-mails of probation officers but they could misplace the e-mail in their spam inbox, for instance. Conducting internet research may have contributed to the fact that the respondents were mainly people familiar with new technology.\n\nAnother limitation could be the lack of in-depth interviews with the probation officers. Such data would allow for a more detailed description of situations which probation officers find especially difficult during the pandemic. This suggests that further studies on probation officers using both qualitative and quantitative measures are warranted.\n\n\nConclusions\n\nThe pandemic has changed social reality. It has changed the economy, revolutionised social systems, including the work of the administration of justice and court-appointed custodians. For persons easily influenced by negative information on social media, the pandemic is a period which intensified problems experienced by these persons, the feeling of uncertainty, fear and sometimes introduced loss and pain for their loved ones.\n\nTherefore, it seems justified to provide quick support to families regarding skills in solving conflicts through mediation, communication training, financial education and access to free-of-charge psychotherapeutic support. For court-appointed custodians the introduction of education among wards regarding availability of institutions, organisations and persons providing the aforementioned support, is an important task.\n\nSince we can expect further increase in the frequency of occurring personal and social problems experienced by court-appointed custodians’ wards, financial resources and energy of social institutions, cooperation with court guardianship should be focused on psycho-educational campaigns concentrating on providing information on symptoms of problems in the mental and emotional sphere. Preventing exclusion of groups at risk in the labour market and supporting families to prevent the escalation of psychopathology, development of addictions and domestic violence should be priority areas.\n\nFurthermore, in order to extend the support provided for wards under the supervision of court-appointed custodians and unburden the court guardianship from the excess of cases, it would be valuable to create support systems in schools and social institutions by providing helplines, online duty hours and consultations. It is also necessary to develop psychoeducational programmes, psychological support, offers of environmental clubrooms, socio-therapeutic centres, mental health and pedagogical-psychological clinics with pandemic-related aspects.\n\n\nData availability\n\nFigshare: data upload.csv. https://doi.org/10.6084/m9.figshare.19447193.v3 (Opora, 2022).\n\nThis project contains the following underlying data:\n\n- Data.xlsx [raw data collected during survey. The columns contain answers and the rows contain cases]\n\nThis project contains the following extended data:\n\n- questionnaire in English.docx\n\n- questionnaire in Polish.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\nAcier D, Kern L: Problematic Internet use: Perceptions of addiction counsellors. Comput. Educ. 2011; 56(4): 983–989. Publisher Full Text\n\nAhmed E, Braithwaite V: Bullying and victimization: cause for concern for both family and schools. Soc. Psychol. Educ. 2004; 7: 35–54. Publisher Full Text\n\nAl’Absi M: Stress and addiction: biological and psychological mechanisms. Amsterdam:Elsevier;2010.\n\nAronson E: Człowiek istota społeczna. Warszawa:PWN;2005.\n\nCarr N: Probation in a pandemic. Probat. J. 2021; 68(1): 3–7. (accessed 09.08.2021). Publisher Full Text\n\nGruber J, Prinstein MJ, Clark LA, et al.: Mental health and clinical psychological science in the time of COVID-19: Challenges, opportunities, and a call to action. Am. Psychol. 2021; 76(3): 409–426. PubMed Abstract | Publisher Full Text\n\nGUS: Informacja o rynku pracy w drugim kwartale 2020 roku (dane wstępne). 2020. (accessed 11.10.2021).Reference Source\n\nHamer H, Baran M: Wpływ pandemii na zachowania, postawy i dobrostan Polaków. Warszawa:SWPS;2021. (accessed 01.10.2021).Reference Source\n\nJanus-Dębska A, Gronkiewicz-Ostaszewska M: Kurator sądowy – zawód szczególnego ryzyka. Bezpieczeństwo kuratorów sądowych w świetle badania ankietowego. Warszawa:Wydawnictwo Instytutu Wymiaru Sprawiedliwości;2019.\n\nThe Ministry of Justice, Department of Strategy and European Funds: Department of Strategy and European Funds. Report on the activity of the probation court service for the year 2019, Warszawa.2020a.\n\nThe Ministry of Justice, Department of Strategy and European Funds: Department of Strategy and European Funds. Report on the activity of the probation court service for the year 2020, Warszawa.2020b.\n\nOpora R: data upload.csv. figshare. Dataset.2022. Publisher Full Text\n\nPratt CT: Addicted to incarceration. London:Sage Publications;2009.\n\nPtaszek G, Stunża GD, Pyżalski J, et al.: Edukacja zdalna: co stało się z uczniami, ich rodzicami i nauczycielami?. Gdańsk:Gdańskie Wydawnictwo Psychologiczne;2020.\n\nPyżalski J: Agresja elektroniczna i cyberbullying jako nowe ryzykowne zachowania młodzieży. Kraków:Wydawnictwo Impuls;2012.\n\nUrban B: Zachowania dewiacyjne młodzieży. Kraków:Wydawnictwo Uniwersytetu Jagiellońskiego;2000.\n\nWhaley A, Snyder C, Kent C, et al.: Presentence Work in COVID – 19 Environment. Federal Probation;2021; Volume 85 Number 1.\n\nVanstone M: Probation and Social Work on Trial. Criminol. Crim. Just. November 2012; 12(5): 617–618. Publisher Full Text"
}
|
[
{
"id": "158433",
"date": "03 Jan 2023",
"name": "Agnieszka Lewicka - Zelent",
"expertise": [
"Reviewer Expertise special pedagogy",
"criminology",
"social rehabilitation",
"mediation"
],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nDespite the removal of restrictions in many countries, COVID-19 continues to take its deadly toll around the world, meaning that it is still present and threatening people. The author of the article has attempted to identify the problems faced by probation officers in Poland during the pandemic. Such findings are important in the context of the possibility of the return of sanitary restrictions resulting in changes in the ways in which probation officers do their work and support their charges. More than 200 probation officers participated in the author's research. Based on the results of the research, recommendations can be made for changes in the preparation of probation officers for their work and in the training of active probation officers. It would be worthwhile to see whether probation officers in other countries need to learn to cope with new difficulties, the effects of COVID-19.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\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": "158436",
"date": "04 Jan 2023",
"name": "Barbara Contreras Montero",
"expertise": [
"Reviewer Expertise Inequality and social exclusion",
"especially homelessness."
],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 article addresses an important issue regarding the perception of probation officers in Poland during COVID-19 about the family situation of wards under supervision. This is especially relevant from the perspective of Social Work, as long as it shows how the quarantine/isolation affected the families and the wards, and therefore the intervention of the probation officers. The strengths of this manuscript are:\nThere is a clear question or objective, which is measured by a number of relevant variables.\n\nThe methodology and sample are adequate.\n\nIn general, the data is well structured and well presented.\n\nThe discussion and conclusions show a relevant analysis of the topic.\n\nSpecific Comments\nHowever, some suggestions are given to improve the manuscript. reorder the document to make it more understandable. It is suggested:\nReview the introduction in order to detect some small errors in English or repetitions of the same word in the same sentence or the next one. For example, both, “video conference” and “videoconferencing” are admitted, but in the text is written “videoconferencing” an “video conferencing” Indistinctly. This needs consistency.\n\nIn the seventh paragraph of the introduction, the first sentence \"Describing the reality...\" is catchable, since it does not provide relevant information and is not entirely linked to what follows.\n\nThe analysis of the results is organized according to table 1. However, there is a small jump after the \"narcotics/drugs use\" and a disorganization in the last variables. It is understood that it is appropriate to comment on the section on “domestic violence” just before talking about other “aggressive behaviors”, but this loses the order of the table. It is recommended to rearrange the items in the table so that they correspond to the order of the analysis.\n\nThere is a small typo/mistake in the comment about \"Family fights\". The table shows 0.46% while the analysis speaks of 0.45%. These numbers must match.\nRecommendation: Approved with only minor changes.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? 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": "158435",
"date": "05 Jan 2023",
"name": "Mary Grace Vella",
"expertise": [
"Reviewer Expertise Criminology",
"Restorative Justice",
"Social Policy"
],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe article aimed to gain a better understanding of how the difficulties faced by clients of the Polish Probation Service have changed during the COVID-19 pandemic, and how this as a result has impacted the nature of work of probation officers. Thus, the aim of the study was to gain further insight into the views and perceptions of probation officers on the challenges encountered in the performance of their duties, with the aim of contributing towards the development of priority foci for future interventions and service provision post-Covid.\n\nThe application of this task was carried out through the adoption of a quantitative survey research by the use of a closed-ended questionnaire. The survey, conducted with 12 randomly selected court regions in Poland among 216 professional court-appointed custodians was sent to all probation officers operating in these regions. This research design was observed to offer a suitable and viable methodological framework for addressing the aims and purposes of the study and for gaining an overview of the transformations shaping the nature of work of Polish probation officers as a result of the pandemic. Due attempt has also been made to secure a cross-sectional representation of the probation workforce through the inclusion and participation of different court regions, to enable generalizability from the results. The study managed to achieve a significantly sized response rate, as from a total of 482 probation officers who received the questionnaire, 216 successfully submitted their responses.\n\nThe first part of the article aptly introduces the aims and objectives of the study and its underlying rationale, through embedding the research problem within the Covid-19 pandemic contextual framework. The chapter demonstrates that the researcher has given ample initial reflection to the nature and purpose of the study, as well as to the overall methodological design and research process. Attention has also been addressed to the development of a suitable research question to focus and guide the conduction of the study. The underlying aim of the study is indeed adequately articulated through a relevant research question, i.e. ‘Have social problems in families under the supervision of a court-appointed custodian intensified during the pandemic?’ However, the paper could have presented a more detailed contextualized exploration of the researched population, by referring to the nature and structure of probation work in Poland, including the legislative framework governing community-based sanctions, the main duties and specialisations, caseload, as well as the main challenges associated with probation work. Such information is particularly relevant for those who are not familiar with the nature and structure of probation work in Poland, and would have provided a more comprehensive analysis of how court appointed supervision work operated pre-pandemic, thus enabling better comparability with the results emerging from the study.\nThe methodology section of the study provides a succinct, yet inclusive overview of the methodological design of the study, including processes of data collection and analysis, as well as ethical considerations adopted for the purpose of the research. The paper demonstrates insight of the methodological strengths and weaknesses of the study and has aptly attempted to mitigate these limitations and biases. A main strength of the study concerns the researcher’s successful attempt to gain response from a large sample of probation officers from all over Poland, specializing in juvenile, adult, as well as family cases. A methodological challenge which could have been further explored, concerns attempt to gain a more representative sample of probation officers in terms of socio-demographic characteristics and expertise, such as through the implementation of a clustered random sampling technique. Complementing the quantitative analysis through rich and in-depth qualitative data arising from interviewing with practicing probation officers would have enabled a more insightful and experiential approach, as well as a more holistic analysis of the issue under investigation. Despite the objective quantitative nature of the study which enables generalisability, recognition could be further made that given the examination of probation officers’ perceptions and experiences, the study is still based on the collation and measurement of subjective responses. The article has aptly put forward relevant recommendations and proposals for further research on the subject matter, including the location of the study within an ongoing wider comparative research project with probation officers from other countries. The article also provides a good overview of the ethical considerations taken on board to mitigate any arising ethical challenges arising from the conduction of the study, which given the anonymous nature of the survey research, were minimal.\nThe findings arising from the survey research with probation officers have been presented in a systematic and orderly manner in both descriptive and tabular form. However, the findings could have highlighted a bit further the divergences, possibly through a compare and contrast approach, between pre- and post-pandemic experiences. The findings of the study were also aptly discussed and analyzed in line with existing contemporary literature in the field. Thus, the article confers a thorough and focused presentation and analysis of the findings as emerging from the results of the survey research.\n\nThe study pursues an interesting and highly relevant topic of analysis for the area of probation work. The research is well-designed and organised and the results arising from the study are aptly discussed and presented in line with important areas arising from the literature. The research has provided added contribution to knowledge not only in terms of the impact of the pandemic on social and probation work in Poland, but also in terms of the wider impact of the pandemic on multidimensional aspects of wellbeing. It is interesting to note that as in various other contexts, such as in the areas of social work and pedagogy, reduced caseload and restrictions posed by the pandemic, did not lead to a reduction in work for probation officers, but the complexity created by the pandemic situation instead led to the exploration for other alternative and innovative solutions.\n\nOn the basis of the research findings, the study has put forward a number of policy recommendations for addressing emerging trends and the amelioration of existing practices. Despite the validity of the proposed priority areas, referring amongst others to; holistic support to families, the consolidation of psycho-educational campaigns, addressing labour market exclusion for at-risk groups, as well as the development of support systems in educational and social institutions as well as various other services and programmes focusing on psychological health and wellbeing, proposals could have been more specifically focussed on addressing the particular challenges of court supervision and probation work. Through such concerted focus on the challenges facing probation work, by for example making reference to the need, if required, for legal reform and underlying philosophy or organisational structure and resources, would have provided more relevant contribution to knowledge by addressing the challenges of probation work in a more holistic and comprehensive manner.\n\nIs the work 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/11-1528
|
https://f1000research.com/articles/11-1526/v1
|
15 Dec 22
|
{
"type": "Research Article",
"title": "Serum leucine-rich alpha-2 glycoprotein levels in rheumatoid arthritis and spondyloarthritis: A promising biomarker",
"authors": [
"Rizqi Arini Siregar",
"Suryo Anggoro Kusumo Wibowo",
"Sumariyono .",
"Aulia Rizka",
"Rudy Hidayat",
"Hamzah Shatri",
"Sukamto Koesnoe",
"Cosphiadi Irawan",
"Suryo Anggoro Kusumo Wibowo",
"Sumariyono .",
"Aulia Rizka",
"Rudy Hidayat",
"Hamzah Shatri",
"Sukamto Koesnoe",
"Cosphiadi Irawan"
],
"abstract": "Background: In the early stages of the disease, some of the signs and symptoms of joint inflammation in rheumatoid arthritis (RA) may resemble that of spondyloarthritis (SpA). An examination that can help distinguish RA and SpA is warranted. One such examination is the measurement of serum leucine-rich alpha-2 glycoprotein (LRG) levels. This study aimed to measure serum LRG levels in RA and SpA patients and determine the role of LRG in the diagnosis of RA and SpA. Methods: This is a cross-sectional study consisting of 26 RA subjects and 26 SpA subjects. The SpA subjects were further grouped into ankylosing spondylitis (AS), psoriatic arthritis (PsA), and peripheral SpA. Measurement of serum LRG levels were conducted using ELISA. Difference between LRG levels of the two groups were compared using the Mann-Whitney test. Results: LRG levels were elevated in 76.9% and 84.6% of subjects with RA and SpA, respectively. The median LRG levels were higher in RA subjects (77.03 (27.16–107.73)) than SpA (68.67 (33.15–115.18)). There was no significant difference in LRG levels in RA and SpA subjects (p = .442). The RA and PsA group were predominated by diseases of moderate activity, 88.5% and 58.3%, respectively. In comparison, AS was dominated by high disease activity (85.7%). The highest median LRG levels in AR and SpA subjects were in new-onset patients (82.21 vs. 72.25 µg/dL). Conclusions: There was no significant difference in LRG levels between RA and SpA subjects. The role of LRG in the diagnosis of RA and SpA remains to be determined in future studies.",
"keywords": [
"rheumatoid arthritis",
"spondyloarthritis",
"leucine-rich alpha-2 glycoprotein"
],
"content": "Introduction\n\nArthritis is an inflammatory process characterized by pain, swelling and stiffness of the joints. Chronic inflammation of the joints can lead to joint deformity and destruction of structures in proximity to the joints. Early identification of the etiologies of arthritis is pivotal to promote clinical improvement, prevent the development of structural and functional joint damage, and consequently preserve patient quality of life.1 Two of the major causes of arthritis are systemic autoimmune diseases, such as rheumatoid arthritis (RA) and spondyloarthritis (SpA).2,3\n\nThe prevalence of these two diseases is similar. The global prevalence of RA is 0.5–1%, while the exact prevalence of RA in Indonesia is unknown. However, based on a prevalence of 0.5–1% of the total population of Indonesia, it can be estimated that RA occurs in around 1.3 million Indonesians in 2020.4 Meanwhile, the prevalence of SpA is about 0.5–2% of the world population.5,6\n\nThe pathogenesis of RA and SpA is complex and distinct from one another. RA is an autoimmune disease characterized by the production of rheumatoid factor (RF) and anti-citrullinating protein antibody (ACPA).7,8 Patients with RA who are positive for RF, ACPA, or both, which represents approximately 75–85% of all patients with RA, are classified as seropositive RA. However, approximately 20% of patients with RA are negative for RF and/or ACPA and are consequently classified as seronegative RA.5 The absence of antibodies in SpA renders it difficult to distinguish from seronegative RA in some cases. SpA is a group of systemic rheumatic diseases characterized by chronic autoinflammation of the innate immune system and is associated with HLA-B27.6,7 SpA is further subdivided into psoriatic arthritis (PsA), ankylosing spondylitis (AS), reactive arthritis, arthritis associated with inflammatory bowel disease (IBD) and non-specific arthritis.9,10\n\nIn the early stages of the disease, some of the signs and symptoms of joint inflammation in RA may resemble that of SpA. This can lead to difficulty in pinpointing a diagnosis, which then affects the extent of joint damage. A cohort study by Combe et al., suggested that early diagnosis is the key to a good outcome in the management of arthritis.11 Despite this urgency, classification criteria and clinical diagnostic standards are often only useful one or two years after disease onset. Additionally, clinicians may not use them consistently and uniformly when classifying early-phase arthritis.11–13\n\nThe annual incidence of early inflammatory arthritis (EIA) ranges from 115 to 271 per 100,000 adults. The annual incidence of undifferentiated arthritis (UA) ranges from 41 to 149 per 100,000 adults. Around 13–54% of UA will progress to RA, whereas 21–87% of UA will persist. Several cohort studies report that early-phase arthritis patients who are referred to rheumatology for further assessment had to wait for 2 to 36 months to be reclassified to a more specific arthritis.14 These holdups can lead to delays in diagnosis and administration of therapy. Several studies have shown that patients with RA diagnosed in more than 12 weeks since the onset of symptoms eventually exhibited more severe joint destruction. Over a course of 10 years, 92% of patients showed decreased functional ability and about 50% required assistance in performing daily activities.15,16\n\nProviding a correct diagnosis will positively impact the management of these patients as some medical therapies for RA may not be effective in SpA. For instance, the B cell targeting drug rituximab is considered effective in RA but has yet to be proven effective in SpA. By contrast, the administration of drugs targeting the interleukin (IL)-12/IL-23 and IL-17 pathways have proven beneficial in PsA, but not in RA.17,18\n\nIn some cases, RA and SpA may show clinical symptoms that are difficult to distinguish from one another. Therefore, an examination to assist the diagnosis of RA and SpA is warranted. One such examination is the measurement of leucine-rich alpha-2 glycoprotein (LRG) levels. LRG is a 50 kDa leucine-rich glycoprotein synthesized in hepatocytes, neutrophils, macrophages, and epithelial cells of inflamed tissues. LRG expression is mostly stimulated by IL-6 or IL-1 but could also be induced by IL-1β, IL-22, and NF-α. Increased pro-inflammatory cytokines, especially IL-6, has been shown to indirectly increase LRG.19–21\n\nStudies assessing LRG levels in the synovial fluid of RA and SpA patients have previously been conducted, with varying results being obtained. Several studies examining LRG levels in synovial fluid, including one by Birkelund et al., reported that synovial fluid proteomics analysis from SpA and RA patients showed striking differences in the amount of innate immune system proteins, especially those from neutrophil granulocytes, including LRG. This protein was found to be more abundant in the RA patient group.22 Furthermore, there are also studies examining the utility of serum LRG levels in differentiating EIA. McArdle et al., analyzed serum protein biomarkers from RA and PsA patients using multiple reaction monitoring (MRM). LRG was recognized as one of the proteins that was able to distinguish RA from PsA, which was found to be significantly increased in RA relative to PsA. This increase in LRG is associated with a relatively higher level of IL-6 in RA compared to SpA, which in turn produced a greater increase in LRG expression.17 Nakajima et al., found that serum LRG levels were also elevated in patients with PsA compared to patients with psoriasis vulgaris.23\n\nCurrently, there are no existing studies comparing LRG values in RA and SpA patients using blood samples. Therefore, we analyzed serum LRG levels in patients with confirmed RA and SpA. The results of this study can hopefully be used as primary data to distinguish RA and SpA in cases where they are difficult to discern and aid in better understanding the role of LRG the diagnosis of RA and SpA.\n\n\nMethods\n\nThis study has received approval from the Research Ethics Committee of Faculty of Medicine Universitas Indonesia and Dr. Cipto Mangunkusumo National Central Public Hospital (KET-197/UN2.F1/ETIK/PPM.00.02/2022) on 28 February 2022. Written informed consent for their participation in the study and publication of the patients’ details was obtained from each of the patients. The patients were anonymized and identified through the medical record number.\n\nThis research was a cross-sectional study conducted at the Rheumatology Polyclinic of Dr. Cipto Mangunkusumo National Central Public Hospital (RSCM) (Jakarta, Indonesia) from March to May of 2022. This study is reported in line with the STROBE guidelines.24 The research subjects were recruited by consecutive sampling. The inclusion criteria included: patients aged over 18 years old; confirmed seropositive active RA patients in accordance with the disease activity score-28 for RA (DAS28); active SpA patients in accordance with the classification criteria of each disease, namely AS disease activity score (ASDAS) for AS and peripheral SpA and disease activity index for PsA (DAPSA) for PsA; and (4) patients able to fill out the consent form and are willing to participate in the study. Patients with other systemic autoimmune diseases, acute infectious diseases, liver disorders, and malignancies were excluded from the study.\n\nThis study also divided the subjects to several subgroups. C-reactive protein (CRP) levels were measured and patients with elevated CRP, defined as greater than 5 mg/dL, were analyzed separately. The LRG levels of both RA and SpA patients were also analyzed separately based on their disease activity and disease onset. Naïve patients, defined as patients with no prior exposure to conventional or disease-modifying anti-rheumatic drugs (DMARD), were categorized as “new onset”. Meanwhile patients who have received DMARD were categorized as “old onset”. Appropriate criteria cut-off points were used according to the corresponding disease in determining disease activity. Determination of sample size can be found as Underlying data.24\n\nBlood samples collected from patients were analyzed for LRG and CRP levels in an ISO-certified laboratory at Universitas Indonesia. To extract the serum, 6 mL of whole blood was drawn and centrifuged (Heraeus Labofuge 200, Thermo Fisher Scientific, Waltham (MA), USA) at 3,000 rpm for 15 minutes and 2,000 rpm for 5 minutes for LRG and CRP, respectively. LRG measurement was performed with Human LRG ELISA Assay Kit (Immuno-Biological Laboratories Co., Ltd., Fujioka, Gunma, Japan) and read with spectrophotometry at 450 nm. Meanwhile, CRP measurement was performed with immunoturbidimetry using cobas c 311 analyzer (Roche Holding AG, Basel, Switzerland).\n\nStatistical analyses of the collected data were processed using IBM SPSS Statistics (RRID:SCR_016479) 24.0 for Mac program (IBM Corp., Armonk (NY), USA). Comparison of LRG levels were analyzed using the parametric independent samples t-test or Mann-Whitney non-parametric test depending on the distribution of the data, which was determined via the Shapiro-Wilk test.\n\n\nResults\n\nUltimately, 26 patients with RA and 26 patients with SpA were included as research subjects, totaling at 52 patients. A flow diagram of subject inclusion can be found as Underlying data.24 Characteristics of the research subjects are shown in Table 1.24\n\n\n\n• Male\n\n\n\n• Female\n\n\n\n• Peripheral SpA\n\n\n\n• Ankylosing spondylitis\n\n\n\n• Psoriatic arthritis\n\n\n\n• Moderate (> 3.2–5.1)\n\n\n\n• Severe (> 5.1)\n\n\n\n• Moderate (15–23, 25–29)\n\n\n\n• Severe (> 28)\n\n\n\n• High (≥ 2.1–3.5)\n\n\n\n• Very high (≥ 3.5)\n\n\n\n• Normal (21–50 μg/dL)\n\n\n\n• Increased (> 50 μg/dL)\n\n\n\n• Non-DMARD\n\n\n\n• DMARD monotherapy\n\n\n\n• Combination of 2 DMARD\n\n\n\n• Combination of 3 DMARD\n\n\n\n• Did not use\n\n\n\n• Methylprednisolone ≤ 4 mg\n\n\n\n• Methylprednisolone > 4 mg\n\n\n\n• NSAID\n\n\n\n• New onset\n\n\n\n• Old onset\n\n\n\n• No comorbidity\n\n\n\n• One comorbidity\n\n\n\n• More than one comorbidity\n\n* Mean was used as the data was normally distributed; RA: rheumatoid arthritis; SpA: spondyloarthritis; DAS28: disease activity score-28 for rheumatoid arthritis; DAPSA: disease activity index for psoriatic arthritis; ASDAS: ankylosing spondylitis disease activity score; LRG: leucine-rich alpha-2 glycoprotein; CRP: C-reactive protein; DMARD: disease-modifying anti-rheumatic drugs; NSAID: non-steroidal anti-inflammatory drugs\n\nLRG levels of the SpA and RA patients were analyzed with the Mann-Whitney test because the subjects obtained were not normally distributed. The test showed no statistical difference between the LRG levels of patients in the RA group (mean rank = 28.12) and those in the SpA group (mean rank = 24.88); Mann-Whitney U = 296, p = .442 two-tailed.\n\nIn this study, patient LRG levels were further grouped based on disease activity, onset, comorbidity, and treatment (Table 2). Meanwhile, Table 3 shows the serum LRG levels of RA and SpA patients based on disease onset. Additional analyses were also conducted to determine the difference in LRG levels between the RA group and the SpA group based on CRP levels and disease activity.\n\nPatients with elevated CRP were analyzed separately as means to control the potential subjectivity in the assessment of disease activity. Elevated CRP was observed in 21 RA patients and 20 SpA patients. As the data were not normally distributed, the Mann-Whitney test was used. The test showed no statistical difference between the LRG levels of patients with RA (mean rank = 23.24) and those with SpA (mean rank = 18.65); Mann-Whitney U = 163, p = .220 two-tailed.\n\nComparison of LRG levels between RA subjects with moderate disease activity (n = 22) and SpA with high-very high disease activity (n = 19) was also conducted. This additional analysis was performed to further clarify the difference in LRG levels between the two largest groups, which was composed mostly of the RA and SpA group. As the data were not normally distributed, the Mann-Whitney test was used. The test showed no statistical difference between the LRG levels of patients with RA of moderate activity (mean rank = 21.82) and those with SpA of high-very high activity (mean rank = 20.05); Mann-Whitney U = 191, n1 = 22 n2 = 19, p = .144 two-tailed.\n\n\nDiscussion\n\nThis study recruited 26 RA patients and 26 SpA patients who visited the RSCM Rheumatology Polyclinic from March to of May 2022 and met the inclusion and exclusion criteria of the study. The sex of the RA subjects was predominantly female, aligning with the theory that women have a 2–3 times higher risk of developing RA than men. However, it was reported that no meaningful difference exists in RA incidence between men and women men over 70 years old.5 There is strong evidence that autoimmunity is influenced by genetics and sexual chromosomes. For instance, estrogen has been shown to increase the secretion of pro-inflammatory cytokines and exert anti-apoptotic activity and its receptors are found on various immune cells. Hence, estrogen is associated with the development of RA.25\n\nThe majority of SpA subjects in this study were female. This predilection was also observed in the SpA subgroups, namely AS and PsA. Previous study by Baumberger et al., had shown that the majority of AS patients are male, with a ratio of 57:1 in 1980 and 1.03:1 in 2016.26 This is also supported by a study conducted by Tsui et al., which had shown that the presence of the tissue non-specific alkaline phosphatase (TNAP) haplotype, which interacts with the progressive ankylosis protein homolog (ANKH) gene (a gene that is involved in osteogenesis and ankylosis in AS), is associated with AS in men.27 Although AS is more dominant in men than women. Another subtype of SpA, namely PsA, affects men and women equally.28–30 Sex distribution is related to disease presentation because men tend to have axial involvement, whereas peripheral joint involvement is more commonly seen in women. Kennedy et al., reported that the ratio of male to female sex in PsA subjects is 2.1–4.8:1. However, it should be noted that the study took many samples with axial involvement, which is more commonly seen in men.31 Meanwhile, Nishina et al., reported a different male-dominated result in peripheral SpA patients with a ratio of 2.3:1.6.32\n\nThe mean age of RA subjects in this study was 49 years old. Other studies show that the highest incidence of RA cases is in the 50–54 year age group.33 The incidence of RA tends to increase with age. The SpA subjects had a mean age of 41 years, which was consistent with the mean age of the SpA subjects reported by Kennedy et al., i.e., under 50 years old.31 The mean age of the AS subjects in this study was 38 years old. In one study, 92% of patients with AS were less than 45 years old at disease onset.34 The mean age of the PsA subjects in this study was 41 years old. Research by Deike et al., showed that most cases of PsA began at 50–59 years old. The incidence of PsA increases with age, peaks before the age of 60 and declines aftewards.35 The age of the sole female peripheral SpA patient in this study was 48 years old, consistent with the findings of de Winter et al., which stated that the median age of patients with peripheral SpA was 48 (36–55) years old.36\n\nIncreased LRG levels were observed in the majority of both RA and SpA subjects. The highest median LRG levels were found in the RA group and the median CRP level was also higher in patients with RA. However, normal LRG results were found in six RA subjects (23.1%) and four SpA subjects (15.4%). The elevated LRG levels is consistent with findings by Birkelund et al., wherein joint fluid analysis in RA and SpA subjects showed striking differences in the number of proteins from the innate immune system, especially those from neutrophil granulocytes, including LRG. This protein was found in higher concentration in the RA patient group.22 Moreover, Nakajima et al., reported that LRG is able to differentiate RA from PsA, as shown by the significant increase in LRG levels in patients with RA compared to those with PsA. The increase in LRG concentration may be associated with higher levels of IL-6 in RA, thus increasing the activity of LRG expression.23\n\nLRG is a protein secreted during the inflammatory process, which like CRP, is also an acute phase protein. In patients with RA, serum LRG levels were correlated with DAS28-ESR score and CRP level.19 From the six RA subjects who had normal LRG levels in this study, two had normal CRP levels. In addition, all RA patients with normal LRG levels were old onset patients who have previously received DMARD. Methotrexate is a DMARD that lowers IL-6, an important cytokine in the formation of LRG.32 The same is true for SpA subjects; from the four subjects with normal LRG levels, there was one subject with normal CRP levels. All subjects classified as old onset had received DMARD therapy in the form of methotrexate and sulfasalazine. Hence, the observed normal CRP levels and the effect of DMARD treatment may explain the presence of subjects with normal LRG levels in this study.\n\nBased on disease onset, five RA subjects were classified as new onset patients, while 21 subjects were classified as old onset patients. LRG levels showed higher results in RA subjects classified as new onset patients. As for the SpA subjects, one subject was categorized as new onset and 25 other subjects were categorized as old onset. Aside from being untreated, the higher level of LRG observed in subjects with new onset disease may be related to IL-6 expression in the early stages of the disease. As suggested by animal studies, synovial tissue samples of animals with antigen-induced arthritis (AIA) and adjuvant-induced arthritis (AA), revealed that the expression of IL-6 peaked in the early stages of the disease and decreased in the later stages of the disease.37 Another study also found that IL-6 is expressed in the sacroiliac joints AS subjects of new disease onset.38\n\nIn this study, 17 RA subjects had comorbidities, among them were type 2 diabetes mellitus (T2DM), osteoarthritis (OA), fibromyalgia, controlled hypertension, and pregnancy of eight weeks. The highest concentrations of LRG were found in RA patients who also had OA and fibromyalgia. Of all the subjects with SpA, 14 had comorbidities, including controlled hypertension, IBD, dyslipidemia, T2DM, osteoporosis, OA, and hyper-IgE syndrome. The highest LRG levels were obtained in subjects without comorbidities as they happen to be new onset patients.\n\nSeveral studies support the prevalence of DM in patients with RA. TNF-α and IL-6 are associated with the pathogenesis of DM, insulin resistance and RA. Long-term exposure to elevated levels of IL-6 triggers insulin resistance, which may play a role in the incidence of DM in patients with RA.39 Plasma LRG levels can predict the progression of kidney disease and the incidence of albuminuria in patients with DM. LRG levels in subjects with albuminuria were significantly higher than subjects with stable albuminuria or those in regression. LRG levels in DM subjects with chronic kidney disease (CKD) progression were also much higher than subjects with normal kidney function.40 In another study, LRG levels were twice as high in subjects with end-stage renal disease (ESRD) who had been treated with hemodialysis than in patients with stage 2/3 CKD.41 Prospective study in Southeast Asia in subjects with T2DM stated that high plasma LRG levels were associated with an increased risk of heart failure events.42 LRG is a novel factor in the pathogenesis of heart failure in patients with T2DM. Elevated serum LRG is also associated with peripheral arterial disease and several risk factors for cardiovascular diseases, such as arterial stiffness and endothelial dysfunction.43 Furthermore, LRG levels were found to be elevated in subjects with IBD, as shown by the elevated levels of IL-6 in the colonic mucosal tissue of patients with IBD.44\n\nLRG levels are also elevated in other inflammatory diseases such as OA. In a murine model of OA, increased LRG expression was found in the subchondral bone and articular cartilage.45 The increase in LRG is also associated with IL-6 as it is one of the pro-inflammatory cytokines that is elevated in OA synovial fluid samples.46 In addition, LRG is also a biomarker of fibromyalgia.23,47,48 As serum IL-6 levels are elevated in subjects with fibromyalgia, it is suggested that fibromyalgia may affect the assessment of inflammatory activity in patients with RA.49 This is in line with the results of this study, which showed that RA subjects with OA and fibromyalgia had the highest concentration of serum LRG.\n\nThe highest levels of LRG in RA subjects were found in subjects who had not received DMARD therapy, aligning with previous studies that showed that DMARD therapy reduced IL-6 concentration in patients with RA.50 As for AS subjects, the highest LRG levels were found in subjects with a combination of DMARD and steroid treatment, while the lowest was in the combination of DMARD and paracetamol. In PsA subjects, the highest LRG levels were found in the DMARD monotherapy group and the lowest in the DMARD and steroid combination group. It can be inferred that DMARDs and steroids play a role in regulating IL-6 and blocking NF-κB as promoters of LRG synthesis.32,50,51\n\nLRG levels were found to be increased in 76.9% of subjects with RA, with the highest median LRG level observed in subjects with RA (77.03 vs. 68.67 μg/mL). These results align with the studies of Fujimoto et al., Naka et al., and Serada et al., which reported the elevation of serum LRG in various autoimmune diseases, including RA.19,52,53 Ha et al., also showed that LRG can differentiate RA patients from normal controls as serum LRG concentrations were significantly increased in patients from the RA group compared with patients in the control group.54 LRG stimulation is influenced by pro-inflammatory cytokines, one of which is IL-6, an inflammatory cytokine that is critically involved in the pathogenesis of RA and significantly correlated with serum LRG levels.55 Serum and synovial IL-6 levels are elevated in RA patients and are associated with disease activity.56 Previous study conducted by Madhok et al., showed that RA patients had higher levels of IL-6 (55 (28–139) IU/mL) compared with the control group (10 (7–12) IU/mL).57\n\nIncreased LRG levels were seen in 84.6% of subjects with SpA, with a median of 68.67 (33.15–115.18) μg/ml. Nakajima et al., previously reported that LRG levels were increased in patients with PsA (51.1 ± 32.9 μg/mL) and psoriasis vulgaris (37.8 ± 25.1 μg/mL) compared with controls (25.5 ± 11.5 μg/mL).23 A multicenter retrospective observational study showed that serum levels of IL-6 are elevated in AS and PsA subjects.38 A previous study also reported that the mean IL-6 level was significantly higher in AS subjects compared with controls.38 The highest median LRG levels in SpA subjects were found in the PsA subset in the present study.\n\nThis study found no significant difference between LRG levels in subjects with RA and SpA. This result is different from a previous study by Lee et al., LRG was also previously identified as a protein that can differentiate RA from PsA as shown by the significantly increased LRG levels in MRM analysis of patients with RA.58 In another study of 32 synovial fluid samples from RA patients and 24 synovial fluid controls, it was found that LRG in RA synovial fluid and SpA correlated significantly with synovial fluid circulating free DNA (cfDNA), and it was known that LRG was closely related to plasma CRP.59\n\nThe insignificant difference in this study may be due to other factors affecting LRG levels, including disease onset and treatment received. However, the effects of DMARD treatment on LRG levels in RA and SpA subjects have not yet been studied. Old onset subjects predominated this study. Disease onset is related to IL-6 concentration. IL-6, a known precursor of LRG, increases in both mild and severe inflammatory conditions. Higher LRG levels in subjects with new-onset may be related to IL-6 expression in the early stages of the disease, as suggested in a study by Ferraccioli et al., which showed that IL-6 is a pro-inflammatory cytokine that is expressed in the early stages of the disease and plays a role in pathogenesis.37 Expression of IL-6 in sacroiliac joint biopsies of AS patients is also associated with new disease onset.38 Besides IL-6 activity, disease onset is also related to the patient's initiation of the treatment regimen. Old onset patients in the present study were defined as patients who had been diagnosed before the time of sampling and had received DMARD. Studies have shown that RA subjects receiving DMARD therapy have decreased serum concentrations of IL-6, a known precursor for LRG formation.60 Most of the research subjects also received DMARD treatment in the form of sulfasalazine; from the results of previous studies, it was known that sulfasalazine caused a decrease in LRG levels in four female albino rats suffering from ulcerative colitis.51 The effect on subjects with SpA and RA is still unknown.\n\nBecause this study initially did not consider the level of disease activity, there was a possibility for the patients’ disease activity to be distributed unevenly. The results of this study found differences in the predominant disease activity between the two groups, wherein the RA group was predominated by moderate disease activity, while the SpA group was predominated by high to very high disease activity. The insignificant difference in LRG levels between the two groups in this study may be due to this distribution of disease activity. The difference in LRG levels between RA and SpA subjects may be more apparent if the disease activity between these two groups were controlled. A previous study conducted by Ha et al., only compared LRG levels between active and inactive RA patients, where serum LRG concentrations were found to be significantly higher in patients with active RA compared with patients with RA in remission.54 No previous study has compared LRG levels in moderate and high disease activity; the said study did not distinguish between disease activity groups. Meanwhile, we found different levels of LRG in the moderate and high to very high activity groups.\n\nMedian LRG levels based on increased CRP levels showed higher results in RA subjects than in SpA. The Mann-Whitney test results showed no significant difference in LRG levels in RA and SpA subjects with increased CRP levels. Previous studies have stated that LRG is correlated with CRP levels, as such a positive correlation between the two variables were expected.55 Future research should confirm this finding.\n\nTo the knowledge of the researchers, this is the first study to compare LRG levels of patients with RA and generalized SpA. The limitation of this study was the different predominant disease activity in the two groups. Future studies with a larger subject population along with control of DMARD status and disease activity is warranted.\n\n\nConclusions\n\nThe median LRG levels of the RA and SpA subjects were 77.03 (27.16–107.73) μg/dL and 68.67 (33.15–115.18) μg/dL, respectively. There was no significant difference in LRG levels between RA and SpA subjects. The role of LRG in the diagnosis of RA and SpA remains to be determined in future studies.",
"appendix": "Data availability\n\nMendeley Data: Underlying data for ‘Serum Leucine-Rich Alpha-2 Glycoprotein Levels in Rheumatoid Arthritis and Spondyloarthritis: A Promising Biomarker’. https://www.doi.org/10.17632/xp52nbs8r3.1. 24\n\nThe project contains the following underlying data:\n\n- Data file 1: Dataset of the study subjects.xlsx\n\n- Supplementary 1: Flow diagram of subject inclusion.docx\n\n- Supplementary 2: Determination of sample size.docx\n\n- Supplementary 3: STROBE Checklist.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\nvan Steenbergen HW , da Silva JAP , Huizinga TWJ, et al.: Preventing progression from arthralgia to arthritis: targeting the right patients. Nat. Rev. Rheumatol. 2018 Jan 9; 14(1): 32–41. 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Publisher Full Text\n\nBaumberger H, Khan M:SAT0417 Gradual progressive change to equal prevalence of ankylosing spondylitis among males and females in switzerland: data from the swiss ankylosing spondylitis society (SVMB). Poster Presentations. BMJ Publishing Group Ltd and European League Against Rheumatism; 2017; p. 929.1-929. Publisher Full Text\n\nTsui HW, Inman RD, Reveille JD, et al.: Association of aTNAP haplotype with ankylosing spondylitis. Arthritis Rheum. 2007 Jan; 56(1): 234–243. PubMed Abstract | Publisher Full Text\n\nLeung Y, Tam L-S, Li EK: The Perspective on Psoriatic Arthritis in Asia. Curr. Rheumatol. Rep. 2011 Aug 7; 13(4): 369–375. PubMed Abstract | Publisher Full Text\n\nToloza SMA, Valle-Oñate R, Espinoza LR: Psoriatic Arthritis in South and Central America. Curr. Rheumatol. Rep. 2011 Aug 7; 13(4): 360–368. PubMed Abstract | Publisher Full Text\n\nSetty AR, Choi HK: Psoriatic arthritis epidemiology. Curr. Rheumatol. Rep. 2007 Dec 7; 9(6): 449–454. Publisher Full Text\n\nKennedy LG, Will R, Calin A: Sex ratio in the spondyloarthropathies and its relationship to phenotypic expression, mode of inheritance and age at onset. J. Rheumatol. 1993 Nov; 20(11): 1900–4. PubMed Abstract\n\nNishina N, Kaneko Y, Kameda H, et al.: Reduction of plasma IL-6 but not TNF-α by methotrexate in patients with early rheumatoid arthritis: a potential biomarker for radiographic progression. Clin. Rheumatol. 2013 Nov 11; 32(11): 1661–1666. Publisher Full Text\n\nSafiri S, Kolahi AA, Hoy D, et al.: Global, regional and national burden of rheumatoid arthritis 1990–2017: a systematic analysis of the Global Burden of Disease study 2017. Ann. Rheum. Dis. 2019 Nov; 78(11): 1463–1471. PubMed Abstract | Publisher Full Text\n\nBoel A, López-Medina C, van der Heijde DMFM , et al.: Age at onset in axial spondyloarthritis around the world: data from the Assessment in SpondyloArthritis international Society Peripheral Involvement in Spondyloarthritis study. Rheumatology. 2022 Apr 11; 61(4): 1468–1475. PubMed Abstract | Publisher Full Text | Free Full Text Reference Source\n\nDeike M, Brinks R, Meller S, et al.: Risk of psoriatic arthritis depending on age: analysis of data from 65 million people on statutory insurance in Germany. RMD Open. 2021 Dec 3; 7(3): e001975. PubMed Abstract | Publisher Full Text | Free Full Text\n\nde Winter JJ , Paramarta JE, de Jong HM , et al.: Peripheral disease contributes significantly to the level of disease activity in axial spondyloarthritis. RMD Open. 2019 Jan 11; 5(1): e000802. PubMed Abstract | Publisher Full Text | Free Full Text\n\nFerraccioli G, Bracci-Laudiero L, Alivernini S, et al.: Interleukin-1β and Interleukin-6 in Arthritis Animal Models: Roles in the Early Phase of Transition from Acute to Chronic Inflammation and Relevance for Human Rheumatoid Arthritis. Mol. Med. 2010 Nov 1; 16(11–12): 552–557. PubMed Abstract | Publisher Full Text | Free Full Text\n\nLekpa F, Poulain C, Wendling D, et al.: Is IL-6 an appropriate target to treat spondyloarthritis patients refractory to anti-TNF therapy? a multicentre retrospective observational study. Arthritis Res. Ther. 2012; 14(2): R53. PubMed Abstract | Publisher Full Text | Free Full Text\n\nBordon Y: IL-6, the resistance fighter. Nat. Rev. Immunol. 2014 May 22; 14(5): 282–283. PubMed Abstract | Publisher Full Text Reference Source\n\nLiu J-J, Pek SLT, Ang K, et al.: Plasma Leucine-Rich α-2-Glycoprotein 1 Predicts Rapid eGFR Decline and Albuminuria Progression in Type 2 Diabetes Mellitus. J. Clin. Endocrinol. Metab. 2017 Oct 1; 102(10): 3683–3691. PubMed Abstract | Publisher Full Text Reference Source\n\nYang F-J, Hsieh C-Y, Shu K-H, et al.: Plasma Leucine-Rich α-2-Glycoprotein 1 Predicts Cardiovascular Disease Risk in End-Stage Renal Disease. Sci. Rep. 2020 Dec 6; 10(1): 5988. PubMed Abstract | Publisher Full Text | Free Full Text Reference Source\n\nLiu J-J, Pek SLT, Wang J, et al.: Association of Plasma Leucine-Rich α-2 Glycoprotein 1, a Modulator of Transforming Growth Factor-β Signaling Pathway, With Incident Heart Failure in Individuals With Type 2 Diabetes. Diabetes Care. 2021 Feb 1; 44(2): 571–577. PubMed Abstract | Publisher Full Text Reference Source\n\nCamilli C, Hoeh AE, De Rossi G, et al.: LRG1: an emerging player in disease pathogenesis. J. Biomed. Sci. 2022 Dec 21; 29(1): 6. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMitsuyama K, Sata M, Tanikawa K: Significance of interleukin-6 in patients with inflammatory bowel disease. Gastroenterol. Jpn. 1991 Feb; 26(1): 20–28. Publisher Full Text\n\nWang Y, Xu J, Zhang X, et al.: TNF-α-induced LRG1 promotes angiogenesis and mesenchymal stem cell migration in the subchondral bone during osteoarthritis. Cell Death Dis. 2017 Mar 30; 8(3): e2715–e2715. PubMed Abstract | Publisher Full Text | Free Full Text Reference Source\n\nAkeson G, Malemud C: A Role for Soluble IL-6 Receptor in Osteoarthritis. J Funct Morphol Kinesiol. 2017 Aug 2; 2(3): 27. PubMed Abstract | Publisher Full Text | Free Full Text Reference Source\n\nHsu W, Han D, Ku W, et al.: Metabolomic and proteomic characterization of sng and pain phenotypes in fibromyalgia. Eur. J. Pain. 2022 Feb 26; 26(2): 445–462. PubMed Abstract | Publisher Full Text | Free Full Text\n\nEl-Mikkawy DME, EL-Sadek MA, El-Badawy MA, et al.: Circulating level of interleukin-6 in relation to body mass indices and lipid profile in Egyptian adults with overweight and obesity. Egypt Rheumatol Rehabil. 2020 Dec 9; 47(1): 7. Publisher Full Text\n\nKawanami T, Kawanami-Iwao H, Takata T, et al.: Comprehensive analysis of protein-expression changes specific to immunoglobulin G4-related disease. Clin. Chim. Acta. 2021 Dec; 523: 45–57. PubMed Abstract | Publisher Full Text Reference Source\n\nStraub RH, Müller-Ladner U, Lichtinger T, et al.: Decrease of interleukin 6 during the first 12 months is a prognostic marker for clinical outcome during 36 months treatment with disease- modifying anti-rheumatic drugs. Rheumatology. 1997 Dec 1; 36(12): 1298–1303. PubMed Abstract | Publisher Full Text\n\nBoshra SA: Sulfasalazine attenuates ulcerative colitis in rats via downregulation of miRNA-31, metalloproteinase-3 and high mobility group box 1. Int J Pharm Pharm Sci. 2021 Aug 1; 74–80. Publisher Full Text Reference Source\n\nNaka T, Fujimoto M: LRG is a novel inflammatory marker clinically useful for the evaluation of disease activity in rheumatoid arthritis and inflammatory bowel disease. Immunol Med. 2018 Apr 3; 41(2): 62–67. PubMed Abstract | Publisher Full Text\n\nSerada S, Fujimoto M, Ogata A, et al.: iTRAQ-based proteomic identification of leucine-rich -2 glycoprotein as a novel inflammatory biomarker in autoimmune diseases. Ann. Rheum. Dis. 2010 Apr 1; 69(4): 770–774. PubMed Abstract | Publisher Full Text\n\nHa Y, Kang E, Lee S, et al.: Serum leucine-rich α2-glycoprotein is a useful biomarker for monitoring disease activity in patients with adult-onset Still’s disease. Scand. J. Rheumatol. 2015 Sep 3; 44(5): 399–403. PubMed Abstract | Publisher Full Text\n\nDerakhshan MH, Dean L, Jones GT, et al.: Predictors of extra-articular manifestations in axial spondyloarthritis and their influence on TNF-inhibitor prescribing patterns: results from the British Society for Rheumatology Biologics Register in Ankylosing Spondylitis. RMD Open. 2020 Jul 8; 6(2): e001206. PubMed Abstract | Publisher Full Text | Free Full Text\n\nUrushima H, Fujimoto M, Mishima T, et al.: Leucine-rich alpha 2 glycoprotein promotes Th17 differentiation and collagen-induced arthritis in mice through enhancement of TGF-β-Smad2 signaling in naïve helper T cells. Arthritis Res. Ther. 2017 Dec 14; 19(1): 137. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMadhok R, Crilly A, Watson J, et al.: Serum interleukin 6 levels in rheumatoid arthritis: correlations with clinical and laboratory indices of disease activity. Ann. Rheum. Dis. 1993 Mar 1; 52(3): 232–234. PubMed Abstract | Publisher Full Text | Free Full Text\n\nLee AYS, Chataway T, Colella AD, et al.: Quantitative Mass Spectrometric Analysis of Autoantibodies as a Paradigm Shift in Autoimmune Serology. Front. Immunol. 2019 Dec 4; 10. PubMed Abstract | Publisher Full Text | Free Full Text\n\nShinzaki S, Matsuoka K, Iijima H, et al.: Leucine-rich Alpha-2 Glycoprotein is a Serum Biomarker of Mucosal Healing in Ulcerative Colitis. J Crohn’s Colitis. 2017 Jan 1; 11(1): 84–91. PubMed Abstract | Publisher Full Text | Free Full Text Reference Source\n\nYoshida Y, Tanaka T: Interleukin 6 and Rheumatoid Arthritis. Biomed. Res. Int. 2014; 2014: 1–12. PubMed Abstract | Publisher Full Text | Free Full Text Reference Source"
}
|
[
{
"id": "218898",
"date": "27 Nov 2023",
"name": "Senem Sas",
"expertise": [
"Reviewer Expertise 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\nA well-written and designed study. There are some problems. The authors must explain:\nThe study group is too small.\nIt is important to standardize the demographic data such as disease duration' and age Did the authors mention this? Also why healthy subjects did not participate in the study?\n\nAlso, pregnant patients have participated in the study. A professional statistical consultant is needed to analyze.\n\nLogistic regression analysis may be needed.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Partly\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nI cannot comment. A qualified statistician is required.\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes",
"responses": []
}
] | 1
|
https://f1000research.com/articles/11-1526
|
https://f1000research.com/articles/11-1525/v1
|
15 Dec 22
|
{
"type": "Research Article",
"title": "Agent-based modelling for the study of shipwreck site formation processes: A theoretical framework and conceptual model.",
"authors": [
"Rodrigo Vega-Sánchez",
"Jorge M. Herrera",
"Jorge M. Herrera"
],
"abstract": "Background Shipwreck site formation processes (SFP), their dynamics and transformation have long interested maritime archaeology from both a historical perspective and heritage management since shipwrecks constitute endangered cultural sites. Major contributions to shipwreck SFP have been made since the 1970s which conceive SFP from a systemic theoretical perspective, notably those of Keith Muckelroy and Martin Gibbs. However, to our view, such a perspective falls short in its capacity to explain and predict the distribution of elements in a shipwreck. In this article, we propose that shipwreck SFP can be understood from the theoretical framework of complex adaptive systems (CAS), where a SFP constitutes a CAS in which nonlinear interactions of natural and cultural factors give rise to the observed seabed distribution of a shipwreck as an emergent phenomenon. Methods From this theoretical framework, we propose agent-based modelling (ABM) as a suitable methodological approach for studying SFP. We show its implementation using the USS Somers, a 19th-century brig of war that sank in 1846 off the port of Veracruz, Mexico, during the Mexican-American War as a case study. The conceptual model was developed from the integration of historical data about the ship’s nautical characteristics and operation, information on the wrecking event from eyewitnesses, as well as modern environmental data. Results We present a conceptual model defining various elements that would constitute the Somers’ ABM. It gives specifics about the characteristics and variables regarding agents, global variables, processes, indicators, degradation and deposition sequences, and user interface. Conclusion The conceptual model served to develop ABM in a simulation platform where historical hypotheses can be tested and various possible scenarios of the SFP can be explored. By contrasting the simulation results with the archaeological record of the shipwreck, ABM would allow maritime archaeologists to postulate more supported and refined interpretations of a shipwreck’s SFP.",
"keywords": [
"Shipwreck",
"site formation processes",
"agent-based modelling",
"simulation",
"complex adaptive systems",
"maritime archaeology",
"cultural heritage",
"Mexican-American War"
],
"content": "1. Introduction\n\nIn maritime archaeology, the study of shipwreck site formation processes (SFP) has been a central topic of interest since the beginning of the discipline and has been undertaken by different scholars, and under different theoretical influences. Many authors have made key contributions to the subject, notably Keith Muckelroy in the 1970s and Martin Gibbs in the first decade of the 21st century.1,2 These authors’ SFP models were conceived from a classical systemic theoretical perspective. A system is considered an “intercommunicating network of attributes or entities forming a complex whole. [Entities’] states or values change sequentially with space, time, or both and consequently the overall system state changes”.3 While this perspective revolutionised maritime archaeological thinking during the last quarter of the 20th century, to our view falls short in its capacity to explain and predict the distribution of elements in a shipwreck.\n\nIn this article, we propose that a step forward can be taken beyond the classical standpoint of systems theory to conceive shipwreck SFP as complex adaptive systems (CAS). In the next section, we argue that SFP indeed show features of CAS, where the interaction of large networks of components, without central control and simple operating rules, gives rise to complex collective behaviour, information processing, and adaptation.4 When conceiving SFP as CAS, using methodological tools that allow simulating their non-linear interactions and emerging properties becomes necessary. One such tool is agent-based modelling (ABM), which is particularly useful when the system is made up of a medium and heterogeneous number of individuals, with complex but local interactions that occur in an environment with many interaction possibilities, such as natural environments.5,6\n\nWe then present a conceptual model for developing an ABM for studying shipwreck SFP, taking the 19th-century brig-of-war USS Somers (1846) as a case study. This research is part of the Proyecto Arqueología Marítima de la Guerra de Intervención (1846-1848) (PAMGI), a maritime archaeology project developed by the Institute of Anthropological Research within the National Autonomous University of Mexico (IIA-UNAM), and with support from UNAM, the British Academy, the Mexican Science Council (Conacyt) and in collaboration with the Centre for Maritime Archaeology, University of Southampton. The project studies the nineteenth-century Mexican-American war not only from a maritime archaeology perspective but also from conflict, historical, and maritime landscape archaeology viewpoints. Although the project works with sites on land, rivers, and seas, a particular emphasis has been devoted to the Somers, from an array of different archaeological and historical perspectives, including SFP.7 By analysing the Somers’ shipwreck SFP through ABM, it is possible to better understand the interactions between various social and natural elements in its formation as an archaeological site.\n\nFinally, we discuss some of the conceptual model’s limitations and potential extensions; and conclude by highlighting our proposal’s theoretical and methodological contribution to archaeology in general, and maritime archaeology in particular.\n\nSFP have been a topic of study in archaeology since the 1970s for understanding “what kinds of intercultural and intracultural variables determine the structure (as distinct from the form and content) of the archaeological record”.8 In maritime archaeology, the early study of shipwreck SFP focused on natural processes to understand the relationship between element disposition and preservation in a shipwreck and its surrounding environment. A three-stage scheme was proposed by Frédéric Dumas for understanding SFP of Mediterranean shipwreck sites, covering from the ship’s sinking to its eventual disintegration and collapse on the seabed.9\n\nDumas’ scheme provided the basis for later models of shipwreck SFP. At the end of the decade, British maritime archaeologist Keith Muckelroy published a seminal model for understanding how it is that “a highly organised and dynamic assemblage of artefacts [the sailing ship] is transformed into a static and disorganised state with long-term stability [the shipwreck site]”.1 Based on the theoretical postulates of David L. Clarke, Muckelroy’s model sought:\n\n“[...] to identify the several features common to any shipwreck [...]. Just as the nature of a ship involves certain basic concepts which are common to all periods and places, so the phenomenon of the shipwreck must involve certain regular features common to all instances. [...] The validity of any conclusions reached in maritime archaeology depends fundamentally on the understanding of these processes, so that their study must occupy a central place in the sub-discipline.”1\n\nIn Muckelroy’s model (Figure 1), shipwreck SFP was conceived as a system in which the ship (input) transforms throughout time by the action of environmental factors (the medium where the wreck took place), extracting filters (those that remove material from the site) and scrambling devices (that rearrange or modify artefacts’ spatial disposition). Thus, the process generates the assembly of artefacts observed on the seabed as output (i.e., the site itself).1 Similar to Dumas’ proposal, a central element in Muckelroy’s model of shipwreck SFP is the surrounding environment, demonstrating the importance of considering the characteristics and dynamics of the maritime environment when studying shipwreck SFP.1 However, to our view, this is a limited conception of the process since the overall element distribution is not predictable from the individual elements and the sub-processes alone. We will delve into this in the next section when discussing complex adaptive systems and emergent phenomena.\n\nFrom Muckelroy, Keith. Maritime archaeology. Copyright Cambridge University Press 1978. Reproduced with permission of the Licensor through PLSclear.\n\nDespite its limitations, Muckelroy’s model represented a milestone in the study of shipwreck SFP. In the following decades, various authors developed and expanded certain aspects of Muckelroy's model. Keith and Simmons extended the concept of “extractor filters” when distinguishing between object removal operations that occurred in antiquity and those of modern times.10 Later, Souza analysed the cultural factors that affect shipwreck distribution, considering pre-depositional processes such as risk minimisation strategies, depositional and post-depositional processes including the intentional abandonment of objects and their subsequent recovery, and priorities in both ancient and modern salvage operations.11 In the early 2000s, O'Shea explored various alternative scenarios based on different types of human intervention, including salvage and search for objects by the original crew in contrast to those carried out by others, and considered the temporal sequence of said activities.12\n\nFor Australian maritime archaeologist Martin Gibbs, Souza and O'Shea's analyses based on the three sub-processes proposed by Muckelroy were still oriented towards explaining the deposition and distribution of the wreck rather than towards the cultural processes involved in the changes suffered by the site through time. Hence, in 2006 he proposed an alternative model for understanding shipwrecks as the result of a cultural process, based on both the nature of the shipwreck event and the sequence and scope of potential responses at each stage.13\n\nGibbs based his model on anthropological studies of human responses to disasters to incorporate the attitudes and responses that people would have before, during, and after the shipwreck into Muckelroy's general scheme (Figure 2). Thus, before the event of the shipwreck itself, not only is the ship considered, but also the actions and decisions that are taken to prevent and avoid the disaster. Immediately before and during the shipwreck, he considers the abandonment and salvage of objects in light of the diverse reactions and interests of the various crew members and passengers. After the disaster, the regrouping and reorganisation of the survivors begins, giving rise to formal rescue actions that can generate independent but associated sites, such as camps and temporary warehouses. Only after all of these social processes have ended that naturally occurring processes, on which traditional research on shipwreck SFP has focused, begin to act on the ship's remains, including sediment deposition or biological colonisation.13\n\nFrom Ref. 13 reproduced with permission from the author.\n\nA decade later, Gibbs himself, along with fellow Australian maritime archaeologist Brad Duncan, supplemented the original model by adding other processes in addition to salvage, that can account for the fate of a ship. These include hulk abandonment and intentional stranding or break-up.2 They also delved into the different motivations that people who carry out different types of rescue may have, which adds to the understanding of the cultural elements involved in SFP.2\n\nIn the last quarter of the 20th century, the field of research that we now call complex systems arose to address the need for studying a wide variety of natural and social phenomena that cannot be understood or explained from the mechanistic and reductionist worldview that has dominated science since the seventeenth century. This field seeks to “[...] explain how large numbers of relatively simple entities organise themselves, without the benefit of any central controller, into a collective whole that creates patterns, uses information, and, in some cases, evolves and learns”.4\n\nAlthough CAS can be very different from each other in their particular details, at an abstract level it is possible to identify certain common properties and thus define them as systems “[...] in which large networks of components with no central control and simple rules of operation give rise to complex collective behaviour, sophisticated information processing, and adaptation via learning or evolution”.4 Furthermore, such collective behaviour “is not predictable from the elements themselves” but arises “through the interaction of the multiple distributed elements”.5 Hence, this type of behaviour is called an emergent phenomenon, which is a characteristic of CAS.\n\nIn both Muckelroy and Gibbs’ models, shipwreck SFP show many of the characteristics of CAS. They involve a series of elements (ship, contemporary actors, environment, future actors), which are subsystems in themselves, whose interactions within the process change over time, generating phenomena that are not predictable from the mere presence of said elements, one of these phenomena being the observable spatial distribution in the wreck. Therefore, in this work we take the theoretical perspective of CAS, particularly on the notion of emergent phenomena, to address shipwreck SFP.\n\nIn maritime archaeology, very few researchers have explicitly adopted the theoretical perspective of CAS. In the late 1990s, Jorge M. Herrera used this perspective to analyse the regional-scale spatial distribution of submerged cultural remains in the keys of Campeche, Mexico.14 Later, together with archaeologists Valerio Buffa and Alejo Cordero (Uruguay) and Jonathan Adams (UK), the first maritime archaeology research program in Uruguay carried out by fully trained maritime archaeologists was designed and implemented from the perspective of CAS, with fieldwork being carried out at both regional and site scales.15 Currently, the theoretical perspective of CAS is a central part of PAMGI.16\n\nIn Germany, Johannes Preiser-Kapeller and colleagues have used the CAS perspective as a theoretical platform to study ancient Mediterranean ports. Preiser developed non-linear models to study the internal dynamics of ports (their establishment, maintenance, and use) and navigation and exchange routes through network models.17 Recently, Rodrigo Ortiz-Vázquez in the UK developed a methodology for the study of wreck site formation processes based on CAS.18 We will give more details about this work in the next section.\n\nIn our view, all SFP can be considered as CAS, the interacting elements within the system being both natural and social, and the observable characteristics of all archaeological contexts as emergent phenomena. In the case of shipwrecks, elements include: the ship, with its specific configuration and a myriad of individual components; the people of the past, those directly or indirectly involved in the design, construction, and operation of the ship with their motivations, beliefs, and decisions; the environment, with its bathymetry, edaphology, currents, storms, temperatures, flora, and fauna (benthic and pelagic); and the people of the future, those who visit the wreck, be it to loot, dive, or study it.\n\nAll these elements make up the CAS constituting agents that interact at one time or another in the SFP. Furthermore, each of them may be a CAS in itself, although on a different scale, thus showing the self-similarity of the process, which is also characteristic of CAS.19 The result of these interactions, changing in magnitude and frequency over time, is an emerging phenomenon: the distribution of elements observable by the archaeologist at the site. But this also changes over time and, in turn, its configuration affects agents and their interactions. For this reason, the configuration of an archaeological site is not a static time capsule or fossil record as Lewis Binford would have suggested,20 but a dynamic one, and it is not predictable from the individual or aggregate characteristics of its composing elements. The shipwreck is more than the sum of its parts; it is a CAS.\n\nOne of the challenges when approaching the study of the emerging phenomena of a CAS is the so-called differential or compositional understanding of the phenomenon. This refers to trying to determine the behaviour of the elements (agents) that gives rise to the pattern that is observed (emergent phenomenon).5 In the case of shipwreck SFP, we try to understand what the characteristics of the elements and the interactions that occurred over time were, which gave rise to the wreck that archaeologists now observe. Shipwreck SFP have relied heavily on the archaeological record derived from survey, excavation, and/or artefact analysis. Different record types generate data of a different nature, amount, and level of detail, and one of the challenges in studying SFP has been integrating such a variety of data to achieve a consistent interpretation of the process.\n\nIn this sense, Rodrigo Ortiz-Vázquez recently developed a methodology for the integration and analysis of information for the study of shipwreck SFP, which explicitly starts from conceptualising them as CAS. Ortiz-Vazquez’s proposal integrates larger-scale information about the site, from historical documents (maps, charts, descriptions), with medium-scale data provided by marine geophysical techniques (multibeam echosounder) and smaller-scale, intra-site data from the photogrammetric record, laser scanning, or excavation.18 By successfully applying his methodology to three case studies, the 18th-century shipwrecks of the Hazardous Prize (1706), the Rooswijk (1740), and the HMS Invincible (1758), Ortiz-Vazquez’s methodology allows analysing a shipwreck’s SFP at different scales, at a three-dimensional level, and over time. This provides much greater detail both to propose solid interpretations of the natural and cultural dynamics that have given rise to wrecks as emerging phenomena, and to their management as cultural resources.18\n\nBut what happens when the historical information and/or the archaeological record of a shipwreck is limited? How can we have such differential understanding and venture interpretations about its SFP when the scarcity of data makes diachronic analyses difficult? How to approach the study of SFP when we only know, for example, some components of the CAS present at the beginning (the ship, the environment) and the emerging phenomenon at the end (the archaeological record of the wreck)?\n\nTo tackle these questions, together with the theoretical framework of CAS, we propose using a methodological approach of computational modelling and simulation as a means of studying shipwreck SFP. We have implemented such an approach to analysing the shipwreck of the 19th-century brig-of-war USS Somers (1846), which we describe further ahead.\n\nVarious types of modelling tools are often used to study CAS and its emerging phenomena, which tend to start from similar principles, but differ in their assumptions and solutions. For example, both equation-based models (EBM) and statistical models assume that the elements of the system are relatively homogeneous with little variability between cases, so they use measures of central tendency to characterise each element.6 However, there are many cases where the system is highly influenced by the heterogeneity of the elements, particularly in social systems. EBM are also usually continuous, not discrete, so they often require relatively large numbers of individuals to function without reaching logically impossible limits, such as having fractions of individuals. Due to these and other limitations, when the system under study is comprised of a relatively medium number of heterogeneous elements, it is better to use discrete models that are closer to natural conditions, such as agent-based models.6\n\nAgent-based modelling (ABM) is a computational tool that allows modelling CAS in order to understand both the rules that configure them and the emerging phenomena that arise from them.\n\n“The core idea of Agent-Based Modeling is that many (if not most) phenomena in the world can be effectively modeled with agents, an environment, and a description of agent-agent and agent-environment interactions. An agent is an autonomous individual or object with particular properties, actions, and possibly goals. The environment is the landscape on which agents interact and can be geometric, network-based, or drawn from real data. The interactions that occur between these agents or with the environment can be quite complex. Agents can interact with other agents or with the environment, and not only can the agent’s interaction behaviors change in time, but so can the strategies used to decide which action to employ at a particular time. These interactions are constituted by the exchange of information. As a result of these interactions, agents can update their internal state or take additional actions”.5\n\nABM is particularly useful when the system under study is composed of a medium and heterogeneous number of individuals, whose interactions are complex but local (depending on the history of the individuals or their individual properties) and occur in a rich environment, i.e., an environment that has many properties or that provides various possibilities for interaction, as in natural geographic environments.6\n\nInterestingly, archaeology was one of the first disciplines in which ABM was applied when this tool was developed at the Santa Fe Institute in the 1990s. It was employed to study the population dynamics of the Anasazi culture that occupied the Long House Valley in Arizona, by comparing the settlement distribution from the simulations with that from the archaeological record.21,22 Since then, ABM has continued to be used as a tool to understand various social processes from the archaeological record, particularly in the areas of prehistoric and hunter-gatherer populations.23\n\nHowever, to date, ABM has not been applied to the study of shipwreck SFP despite the fact that, as aforementioned, it is possible to observe all the features that characterise a CAS in them. To our knowledge, computer simulations have only been used in shipwreck SFP to understand its fluid dynamics aspect. Using a computational fluid dynamics model based on the topography of a shipwreck, sediment, and stream flow data, Quinn and colleagues have shown how the interaction of a wreck with ocean currents and the seabed where it was deposited results in the formation of different types of scouring signatures.24,25 However, their studies use only fluid dynamics models, not ABM, as a simulation tool.\n\nThe Mexican-American War took place between 1846 and 1848, defining the territory, politics, and economic powers of Mexico and the United States (not to say the entire American continent) for the remainder of the 19th-century, with consequences visible to this day.16 A crucial part of US strategy during the War involved blockading Mexican ports, particularly Veracruz. These actions were commissioned to the Home Squadron, the US Navy fleet assigned to the Gulf of Mexico and the Caribbean. One of the vessels in the Squadron was the USS Somers (Figure 3), a brig-of-war 100 feet (30 m) long, 25 feet (7.6 m) wide, and with a hold depth of 11 feet (3.35 m), propelled entirely by sail and armed with ten 32-pounder carronades.26\n\nThe U.S. Brig-of-War “Somers”. Lithography, ca. 1850.34\n\nOn 8th December 1846, while chasing a ship that was trying to break the blockade of Veracruz, the Somers was surprised by a strong wind, capsized, and rapidly sank.27 The general bearings of the Somers’ wreck site, but not its precise location, were known since the mid-nineteenth century thanks to the accounts of some of the survivors.28,29 However, it was not until the mid-1980s that the Somers shipwreck was discovered and, during the 1990s, archaeologically recorded through a collaboration between the Mexican and US governments.14,30 Since then, the Somers shipwreck has only recently been the subject of formal archaeological studies through PAMGI.16\n\nThe archaeological study of the Somers shipwreck represents an excellent case study for conflict maritime archaeology and particularly for the study of shipwreck SFP. Although we know from historical sources the ship’s structural characteristics and the general circumstances that led to its sinking and the wreck’s element distribution after its nonintrusive archaeological recording, we do not know the series of events and conditions, both social and natural, that led to the shipwreck’s current state. That is, we do not know the SFP of the Somers shipwreck. The ABM we describe here was developed to study such SFP.\n\n\n2. Methods\n\nBroadly speaking, the creation of an ABM involves three steps, which are not necessarily sequential but will naturally be modified in a process of feedback and adaptation (constituting a CAS in itself).31 Taking the research question as a starting point, the first step is to create a conceptual model in which all the details that will comprise the ABM are defined, including agents’ characteristics and properties, processes, indicators, and user interface features. It is useful to write these details in the form of pseudocode. This is “a midway point between natural language and formal programming language [that] can be read by anyone, regardless of his or her programming knowledge, while, at the same time containing algorithmic structure that makes it easier to implement directly into real code”.5 Writing the different elements of the conceptual model as pseudocode will facilitate their further programming. It is also convenient to consider from this step the inclusion of elements for model verification, that is, tools that allow easy verification that all the components and processes in the ABM’s simulation platform correspond to what is stipulated in the conceptual model.\n\nThe second step consists of programming the ABM code based on what was defined in the conceptual model. Once programmed, the ABM can be used to explore the characteristics of the CAS that has been modelled and to test hypotheses about it. The third step in creating an ABM is its validation, that is, checking that its elements or behaviours correspond to those of the real phenomenon that has been modelled, regarding those aspects important for our research questions. Validation can be of two types: micro validation, when the behaviours of the ABM’s agents correspond to those of real agents; or macro validation when the emergent properties correspond to real ones.5 Macro validation is the one of interest for our purposes since, as mentioned before, we consider the distribution of elements in the shipwreck as an emergent property. In particular, we would rely on a type of macro validation called empirical validation, where the data produced by the ABM must correspond to the empirical data derived from the observation of real phenomena, i.e., the archaeological record.5\n\nGiven that an ABM of the Somers shipwreck SFP would naturally require a three-dimensional model of the ship, we consulted historical sources that would provide information on the ship’s nautical characteristics, crew, cargo, and artillery. This information allowed us to create a very detailed 3D model of the ship considering its structural elements and other features that, although not part of the ship as such, would have been present within it at the time of its sinking. Sources also included those that gave an account of the shipwreck and possible salvage operations. This information made it possible to adapt both the 3D model and the ABM processes, as we will detail later.\n\nTo model the nautical characteristics of the Somers, we first created a two-dimensional model from a reproduction of the original line plans of the ship published by the nautical historian Howard I. Chapelle in his The History of the American sailing Navy: the ships and their development (Bonanza, 1949, Figure 4). The 2D model contains precise details on the shape, dimension, and position of many of the individual elements of the ship's structure (hull, decks, gunwale), as well as its accessories (windlass, carronades, bilge pump, and stove), and its rigging (masts and sails). We then used this 2D model to create the 3D model that would be used in the ABM (Figure 5).\n\nReprinted from The History of the American Sailing Navy: The Ships and Their Development by Howard I. Chapelle. Copyright © 1949 by W. W. Norton & Company, Inc. Used with permission of the publisher, W. W. Norton & Company, Inc. All rights reserved.\n\nHowever, both Chapelle's plans and the 2D model created from them have some limitations, for obtaining a detailed three-dimensional reconstruction of the Somers. Specifically, a series of structural and accessory elements do not appear in these sources despite constituting fundamental parts of the ship. Some include the frames, metal lining covering the bottom of the hull and rudder blade, stanchions that supported both covers, knees, beams that supported the lower deck, chains to hoist the anchors, and the anchors themselves. Several of these elements or parts of them, particularly the metallic ones (sheathing, chains, and anchors), are indeed found in the Somers shipwreck, so it was essential to take them into account when building the 3D model.\n\nTo construct these elements, we resorted to various historical sources. For the material and dimensions of the metallic sheathing covering the hull and rudder blade, we consulted Chapelle’s book Appendix “General instructions for building a Sloop of War …” and Timoteo O’Scanlan’s Cartilla Práctica de Construcción Naval.26,32 From these sources, sheathing may have been made of brass or copper alloys and reached the uppermost of the ship’s water lines.\n\nAnchors were probably of the “Old Pattern Admiralty Long Shank” type, adopted by the British navy at the end of the 18th century and in use for much of the 19th century33; it is likely that US Navy ships were also equipped with them. Initially, these anchors had a square wooden stock and a ring at the end of the shank. Later, these elements were replaced by a tubular metal stock and a shackle, respectively.33 The Somers was most likely equipped with metal stock anchors; however, not having the precise historical data, we reconstructed them as wooden stock anchors in the 3D model, since this type is the one that appears in the ship’s illustration by Nathaniel Currier34 (Figure 3).\n\nThe chains used for hoisting the anchors are a very noticeable element in the Somers shipwreck, running through the centre from bow to stern covering almost half of the site. To determine their length in the 3D model, we referred to an article by naval historian John H. Harland, stating that it “amounted to three times the depth by soundings, at the very least 25 fathoms or a quarter of a cable, sufficient at all events for the anchor to bite the ground and leave sufficient slack to allow the vessel to drift back far enough to bring the cable nearly horizontal”.35\n\nFor the stanchions’ dimensions, we referred to the appendix of Chappelle's book and for their location on those of the USS Constellation (1854). The knees’ shape and location were also based on the Constellation (Figure 6).\n\nLeft: straight knee supporting the main deck. Right: diagonal knees supporting the lower deck\n\nAdapted from captmoonbeam. USS Constellation at Baltimore Inner Harbor GoPro.\n\nRegarding the type of ballast carried by the Somers, nothing is mentioned in the records we consulted, appearing in neither Chapelle's appendix nor in A. F. Creuze’s Treatise on the Theory and Practice of Naval Architecture (1841).36 However, other US ships of the time, such as the corvettes Vandalia (1828) and Marion (1839) or the submersible H.L. Hunley (1863), carried pig iron as ballast,37–39 so it is reasonable to assume that this would also be the case for the Somers. The Constellation, a 1,268-ton frigate,40 carried 22,940 pounds of wrought iron as ballast.41 Assuming a very similar ratio of ballast to ship size, since Somers was 259 tons, it would have carried around 4,686 pounds of ballast, encompassing a volume of 9.68 ft3 (wrought iron density = 484 pounds/ft3), distributed along the centre of the hold.\n\nDetails about the Somers' artillery, crew and cargo served to add to the 3D model a number of items that are not on the line plans but were certainly on the ship and therefore would affect the shipwreck SFP. Regarding artillery, the Somers was armed with ten carronades, a type of “short cannon of lightweight and large calibre mounted on a slide and on a shaft on which it rotates vertically”.42 These types of cast iron pieces were designed for powerful short-range attacks aimed at decimating both the target ship and its crew14 (Figure 7).\n\nRegarding the crew, line plans indicate three spaces for accommodation and meeting areas for officers: cabins, wardroom, and steerage. Sailors were housed on the same deck as the officers, although not in cabins but in the space between the aft officers' cabins and the fore storeroom. The anchor chains should also have been located in this same area. Regarding the distribution and use of spaces inside the Somers, a sailor who visited the ship at the beginning of 184343 mentioned in a newspaper article that there was\n\n“[...] nothing to make the officers’ quarters but a long trunk house, or companion, raised a few feet from the deck, to let light and air in below, such as you may have seen in our smaller packets which ply along the sea board. [...] The officers’ quarters and the cabin are on the same floor with the berth deck of the crew, separated only by bulkheads, [...] the hold might be so occupied by stores, ammunition, ballast, and the numerous necessaries of a ship of war in actual service …”\n\nThe line plans also show three storage areas: the hold located on the bottom of the hull, which included the magazine; the fore storeroom on the lower deck, which served as a store for spare tools and equipment; and a small area behind the captain's cabin, although it is not clear if this space actually served as a store since its dimensions were quite small (Figure 8).\n\nRegarding the cargo that the Somers would have carried at the time of sinking, the main historical source that would have allowed us to know the details in this regard, the ship's log, was obviously lost during the wreck. For this reason, the closest possible approximation to these details was based on secondary information provided by other social actors present during the blockade of Veracruz in 1846. All information on the movements of the fleet used to be recorded in the ships' logs, particularly in those of the flagships from which Commodores Conner and Perry dispatched, the frigate Raritan and the steamer Mississippi, respectively. As part of PAMGI’s activities in 2019, the logs of both ships were obtained from the US National Archives. The 12th August 1846 entry of the Mississippi log details the number of provisions sent on board the Somers on that day.44 Additionally, the description of the Somers’ wreck written by ship's doctor John H. Wright, originally published in The Daily Picayune newspaper in New Orleans on 22nd December 1846, also mentions some of the provisions on board the ship at the moment of its sinking. From these data, we estimate that in mid-August 1846 the Somers had in the cargo area at least 25 barrels, 5 boxes, 2 kegs, and 40 gallons of provisions; in addition to 140 fathoms (approximately 234 metres) of cables. Although these elements were not part of the ship’s structure, they were also inside when it sank and must therefore be taken into account as part of the shipwreck SFP.\n\nWe consulted various US and Mexican newspapers of the time that reported about the sinking of the Somers or subsequent events related to the survivors. Among those reviewed, the main sources of information were two first-hand accounts of the shipwreck. The first was sent by Captain Semmes to Commodore Perry two days after the accident, who in turn forwarded it to Secretary of the Navy John Y. Mason. The correspondence was published on 2nd January 1847, in the Washington D.C. newspaper Daily National Intelligencer.29 The second, longer, account was the one already mentioned written by the ship's doctor Wright, first published in the New Orleans Daily Picayune28 and republished later in other newspapers.\n\nIn these documents, we looked for mentions about:\n\n1. Parts of the ship or accessories present during its operation and sinking process that may have been removed either intentionally or as a result of the sinking, before reaching the seabed. In Muckelroy’s model, this corresponds to material floated away as a result of the wrecking process. In Gibbs’ model, these types of removal actions are found in the pre-impact warning and impact phases.\n\n2. Salvage operations of objects from the wreck. In Muckelroy’s model, they correspond to the salvage operations sub-process.1 In Gibbs’ model, these types of operations, whether opportunistic or systematic, are found in the rescue and post-disaster phase.13\n\nRegarding the first point, in the accounts of the accident given by Captain Semmes and Doctor Wright, there is no mention of jettison, i.e., heavy objects being thrown overboard, as part of the actions taken in the pre-impact alarm phase. Instead, these sources do report actions taken during the impact phase that would have involved the removal of objects during the sinking process. These actions were:\n\n• attempts were made to loosen the sails, and cut rigging and masts, but without success\n\n• some boxes and hatches or skylights were lost\n\n• the boat was used\n\nThis implies that the 3D model of the Somers used for the ABM should:\n\n• include all major and minor structural components of the ship\n\n• include all the barrels mentioned above, located in the cargo area\n\n• not include the boat\n\n\n3. Results\n\nAlthough the original theoretical models proposed by Muckelroy and Gibbs have been discussed and extended by other authors, we decided to use them as a starting point on which to base the conceptual model for the Somers ABM we present here. In subsequent works, this model will have to be extended to contain other elements and theoretical proposals as necessary, as we will discuss further on.\n\nAs aforementioned, a conceptual model defines in detail the various elements that will make up the ABM: agents and individual properties, procedures (rules of action and interaction of the agents), as well as indicators used as output variables to analyse the model’s results. Both Muckelroy and Gibbs’ models of shipwreck SFP naturally have in common the agents involved and the environment in which they interact but differ in the number and type of sub-processes involved and, therefore, in how these sub-processes affect the objects within the shipwreck. The different elements that make up our conceptual model can be generally grouped into 1) agents, 2) global variables, 3) processes, 4) indicators, 5) degradation and deposition sequences, and 6) user interface. We describe each of them in the following sections.\n\nAn agent can be defined as any “autonomous individual or object with particular properties, actions, and possibly goals”.5 Agents interact in a simulation environment in an adaptive manner, since their interactions can change as a function of time and/or the exchange of information. This results in agents being able to update their internal condition or take additional actions. Given the characteristics of the phenomenon under study, we consider that the best way to approach it would be to develop the ABM in a 3D format so that all the agents have X, Y, and Z coordinates.\n\nOur ABM is comprised of two groups of agents. “Static” agents are those that simulate the environment, including water and the seabed, meanwhile “dynamic” agents simulate the elements of the ship, sediment, and marine organisms. It should be clarified that this classification does not refer to the nature of the agents in an archaeological context, since the marine environment is obviously very dynamic. It merely refers to the behaviour of the agents within the ABM or simulation platform, since both the volume that would surround the ship (representing the water) and the one representing the seabed, do not move. All agents, whether static or dynamic, have common characteristics including spatial location and scale. However, according to the nature of the objects they represent, they also have individual attributes which are detailed later.\n\nAnother type of agent is also present in the ABM, one we could call the “invisible agents”. These are all the people who interacted at some point in the SFP process through their actions and decisions. Although these agents would not appear in the simulations, they are represented in the ABM through the effects of said actions, which are included as options for the initial configuration of the simulation process (see the User interface section below).\n\nRegarding dynamic agents, those that share common characteristics can (and should) be distinguished and grouped in the ABM as families or classes, using a hierarchical system based on shared attributes and procedures. This type of hierarchy and categorisation greatly simplifies programming the ABM and makes it more efficient. Once the general classes of agents have been defined, the individual agents corresponding to each one are generated, inheriting the attributes and procedures of their class while specific attributes and procedures can be assigned to each individual agent. Thus, in the ABM, we use the following classes to distinguish the groups of dynamic agents to be included in the simulation. The first class is the agents that make up the ship, whose class corresponds to the material with which the agent they represent was made: wood-oak, wood-pine, metal-iron, metal-brass, and mixed, the latter being those with combinations of materials such as oak-iron and pine-brass. Later we specify how we defined the materials for each piece and object of the ship. Additionally, there are the sediment and “coral” classes of agents.\n\nAll agents, both static and dynamic, have density and mass as common attributes. Density is assigned according to the material the agent is supposed to be made of (wood, metal, sediment, or coral). Meanwhile mass results from multiplying the assigned density by the agent’s volume. The following sections detail how these, and other individual attributes, are specifically assigned according to the elements they will represent.\n\nSea water is represented in the ABM by the entire 3D environment. It includes attributes of salinity and density that affect various processes; these attributes are defined as global variables to which all agents have access (see Global variables section). The seabed is located at the bottom of the “world”, simulating the natural context where the ship was deposited immediately after sinking.\n\nIn the Somers case, our ABM starts with the ship already settled on the seabed, that is, the model does not take into account the transit from the water surface to the bottom. Although this transit could certainly have altered the distribution of elements of the ship, in this first version of the model we assume that such alteration was minimal and only affected some minor accessory elements, not the structural elements and major accessories (equipment, artillery). In this sense, we assume that what could most alter a ship’s disposition in the transit from surface to bottom would be the listing process, which is considered in the initial conditions of the simulation and detailed later.\n\nAmong the data that has been produced by PAMGI, we have a highly detailed bathymetric survey of the site, produced with a multibeam sonar unit. However, since the postprocessing of this data is currently being undertaken, for the ABM process we assumed a completely flat seabed, without slope. It is very likely that this assumption is not entirely true since the shipwreck site is located a few hundred metres from a reef, so there is surely a certain slope; however, this is barely noticeable when diving at the site. Even so, the slope is an important aspect to consider in the SFP, since it will affect both the dynamics and interactions of natural and cultural elements, as well as the site’s element distribution.1 Therefore, this sea bottom setting will need to be modified in a future version of the ABM based on site-specific bathymetry.\n\nTo simulate bottom sediment movement and allow for the formation of scour pits, the bottom surface should create a random number of individual sediment-class agents. Scour pits are depressions in the seafloor that result from sediment being eroded by waves and currents after the introduction of an object to the seabed (e.g., a shipwreck).24 Sediment attributes are described in Sediment section. The effective formation of scour pits would serve to verify the proper functioning of the ABM, particularly the processes controlling the movement of sediments and currents. In this sense, the appearance of scour pits itself could be considered another emergent phenomenon of the CAS, which arises from the interaction between its mobile (sediment, dominant current, tides) and fixed (ship) elements.\n\nAs aforementioned, we used the 2D model of the Somers as a template for creating a 3D model. For some elements that do not appear in the 2D model, we used other historical documents or images from extant historical ships, such as the USS Constellation. In all cases, the layers of the 2D model or images of the historical documents were imported into Rhinoceros 6 (Robert McNeel & Associates; RRID:SCR_014339) and from each two-dimensional element shown in them, we built three-dimensional objects (Figure 9). This resulted in a 3D model of each element of the ship. The complete 3D model of the Somers can be viewed at https://skfb.ly/o76r8.\n\nMost elements were created from the 2D model of the ship’s lines plans, e.g., the hull’s frames (top).\n\nOthers were based on historical documents (e.g., the helm, centre), or parts of extant historical ships, such as the masts’ fittings on the USS Constellation (bottom).\n\nTo each of the ship’s constituting elements (agents), we assigned the attributes: piece, density, mass, element-category, salvage-value, salvage-difficulty, and degradation-percentage.\n\nThe piece attribute is merely used to identify each of the ship’s elements and to access them individually. As mentioned before, we assigned the density attribute according to the material they represent. In 19th-century sailing ships, different types of wood were used for different parts depending on the required structural needs. Harder woods, such as oak, were generally used in the parts of the ship more exposed to impacts or those withstanding greater structural stress, such as the keel, stem, sternpost, or frames.26 Parts subject to relatively less stress, such as linings or covers, were made of softer woods like pine. For the ABM, the agents’ density value should ideally be assigned according to the specific type of wood from which the pieces they represent were made. However, in the case of the Somers, we currently do not have specific information on these construction details.\n\nThe closest shipbuilding material details to the Somers we have found come from Chapelle’s book Appendix, containing the specifications for the construction of the sloops-of-war Warren and Falmouth.26 Oak wood is specified for the different pieces of the bottom of the hull, bow, and stern, and pine wood for the remaining structural elements of the ship. Based on these data, in the ABM we assigned the density values of wood agents as follows:\n\n• Agents representing pieces of the bottom (keel, false keel, keelson), the bow (stern foot, stem, counter stem, cutwater), stern post, rudder blade, frames, beams, stanchions, and knees were considered to be made of American white oak (live oak), which has a density of 770 kg/m3.\n\n• Agents representing decks, masts, yards, external and internal planking of the hull, escutcheon, and all minor structural elements, were considered to be made of yellow pine wood (heart pine), which has a density of 420 kg/m3.\n\nHowever, since wood density increases when becoming saturated with water, these values should be higher in the simulation. Although we do not have precise data on density change in oak and pine, a recent study with acacia wood showed that, when saturated with water, its density is 8-10% higher than when dry.45 Based on this data, for the ABM we considered that wood is already saturated at the beginning of the simulation, so we increased the density value of agents representing wood by 10%. Thus, agents simulating oak have a density of 847 kg/m3, and those simulating pine of 462 kg/m3.\n\nRegarding metallic elements, we don't have specific data on the Somers. However, in his Treatise on the Theory and Practice of Naval Architecture (1841), A. F. Creuze mentions that on 19th-century ships the fittings and trim were generally made of iron and sometimes copper. The nails used to fasten the linings were usually made of copper, zinc, or tin alloy.36 Timoteo O'Scanlan also mentions in his Diccionario Marítimo Español (1831) that the straps (rings to hold various pieces together as in the masts) are made of iron.42 Finally, Chapelle’s Appendix mentions that the chains carried by Warren and Falmouth were made of iron and the hull sheathing was made of copper.\n\nConversely, Creuze’s mention that the nails were not made of pure copper but of an alloy of zinc, copper, and tin36 may suggest the use of naval brass. Therefore, for ABM we assumed that the latter was used for the hull sheathing. Additionally, the Somers was armed with 32-inch carronades. Both these artillery pieces and the bullets they fired were made of cast iron.14\n\nConsidering the above, for metal agents, we assigned the following materials and densities:\n\n• Minor pieces (fittings and mouldings), chains, anchors, and carronades are cast iron with a density of 7800 kg/m3.\n\n• Hull sheathing is naval brass with a density of 8410 kg/m3.\n\nThe mass attribute of each agent is calculated at the beginning of the simulation from its density and volume. In case the simulation platform to be used does not have an integrated physics module, the mass would have to be calculated with the equation, m=δ×V, where m is mass in kilograms, δ is density in kg/m3 and V is the volume in m3.\n\nFor both the Muckelroy and Gibbs models, it is important to characterise the different ship components in terms of the ease with which they can be removed, and the value imputed to them. These two characteristics are directly related to the removal process, by flotation or salvage operations, and to the rearrangement process by current movement. For example, in the event of a warship sinking, contemporaneous salvage operations would be more likely to be undertaken for recovering artillery, ammunition, or anchors than toolboxes. Similarly, it would be easier to try to salvage minor structural elements or accessories, such as anchors, than major structural elements, such as beams.\n\nFor characterising elements in the Somers 3D model, we based our characterisation on that proposed by Gibbs in table 1 of his 2006 article.13 This element characterisation was the basis for assigning the values of element-category. The latter is a nominal categorical variable with four possible values: 1 = cargo, 2 = fixtures, 3 = minor structural, and 4 = major structural. Element-category, together with the material, was the basis for assigning the values of another two attributes: salvage-value and salvage-difficulty. The values assigned to these attributes for each element of the 3D model can be found in Table 1.\n\nThe salvage-value attribute is a continuous numerical variable whose initial values were assigned to each agent arbitrarily. We assumed that, similar to the previous example, artillery, ammunition, or some equipment would be more valuable for salvaging than structural elements. However, they may be modified at any time. Values could be 1 representing a “low” salvage value, 2 a “medium” value, and 3 a “high” value.\n\nThe salvage-difficulty attribute is also continuous and numerical. To assign the initial values, we made the following assumptions:\n\n• It would be easier to salvage mobile elements than structural elements of the ship. Therefore, mobile elements have lower salvage-difficulty than structural elements.\n\n• It would be easier to salvage items higher up (closer to the surface) than lower down. Therefore, elements with a lower Z coordinate value have less salvage-difficulty than those with a higher Z coordinate value.\n\n• It would be easier to salvage light items than heavy ones. Therefore, salvage-difficulty is also a function of the object’s mass.\n\nThus, the initial value of each agent’s salvage-difficulty is given by the following equation:\n\nAlthough initial values are assigned to the salvage-value and salvage-difficulty attributes at the beginning of the ABM, these values change as the simulation advances, depending on the degradation-percentage of each element and time. This is later detailed in the Execution section.\n\nThe degradation-percentage attribute is a continuous variable. Its initial value set to a random number between 0 and 10% for woods, and between 0 and 5% for metals. This adds stochasticity to the ABM by simulating that the pieces, due to their use, did not have complete integrity at the time of the shipwreck. Each element’s degradation-percentage increases with the simulation time according to the rules established in the Degrade sub-process. In the case of agents representing wood, when this attribute reaches a value of 100, the agent disappears from the environment, simulating its complete degradation.\n\nAgents representing sediment are added to the ABM at two points: 1) as part of the seafloor surface; 2) as part of the ocean current simulation process. These agents represent any type of sediment that is deposited on the site.\n\nSimilar to the bathymetric context, the ABM was designed in a speculative way regarding the sedimentology since we lack data about the specific sediment composition where the Somers was deposited after sinking (near Isla Verde reef) or the sediment that has been covering the wreck since then. This sedimentological characterisation will be part of the next activities by PAMGI. The closest information we have found about such sedimentary composition refers to the Hornos Reef, also in Veracruz. Between 1 and 2 m deep, the sediment is composed of fine (0.125 mm) to coarse (1 mm) sand.46 However, this data cannot be taken as similar to that of the Somers wreck for two reasons: 1) Hornos Reef is located four kilometres northwest of Isla Verde; 2) the Somers shipwreck is 30 metres deeper than the site where the Hornos Reef samples were taken.\n\nIn the coming months, PAMGI will conduct a very detailed marine-geological survey at the Somers’ site to provide us with enough data to produce a comprehensive understanding of the sedimentary processes over time for this specific shipwreck. A future version of the ABM will also include real data from the context, allowing us to produce more realistic simulations, contrast both model versions, and produce a deeper understanding of the ABM’s powers and limitations.\n\nTherefore, for this first version of the ABM, we randomly assigned the value of the agents’ size attribute representing sediment, between 0.00002 and 0.002. Since the unit of distance in the ABM is the metre, these values mean that the smallest particle size is 2 microns and the largest is 2 mm. This random assignment results in a proportional size distribution that represents a silt-sandy sediment (sand = 2.0–0.05 mm; silt = 0.05–0.002 mm).47,48 Considering this type of sediment, we assigned 1265 kg/m3 (silty sand) as the density attribute of these agents. These values, like all others in the ABM, can be later modified when specific data on the sedimentary composition of the Somers shipwreck become available.\n\nBiological colonisation is very common in marine archaeological contexts, particularly in those with metallic elements that serve as support for colonising organisms. However, this colonisation differentially affects metals, being abundant, for example, in iron objects and almost non-existent in those made of bronze. In contrast, submerged wood can also be subject to colonisation, but this is not usually observed in submerged archaeological contexts since the degradation of said material by boring organisms is usually faster than the growth of colonisers.49\n\nCharacterisation of the colonising organisms in the Somers shipwreck has not yet been carried out; this is also one of the next PAMGI activities. However, it is possible to appreciate the great variety of marine flora and fauna in the wreck (Figure 10). In a study on biological colonisation where iron and brass discs were submerged in a similar environment, the waters of Campeche, Mexico, authors found 53 different species on the surface of the discs. Colonisers fell into five categories: 1) organisms with vertical growth, including filamentous macroalgae and bryozoans; 2) encrusting organisms such as sponges, particularly abundant on the iron surface; 3) calcareous tube-forming organisms such as polychaetes; 4) bivalves such as oysters; 5) conical exoskeleton organisms, such as barnacles.50\n\nEven in such a small space (approximately half a metre), the great variety of biological colonisation can be appreciated. Photo: Emilio Vélez Quintero, 2018, courtesy of the author.\n\nThe ABM includes only one type of agent that represents colonising organisms. We collectively named these “corals” and their initial-size is 0.0005. This corresponds to 0.5 mm since the “size of settling larvae usually does not exceed 1 to 2 mm and often is below 0.5 mm”.51 A study on the biomass of different species of colonising organisms sampled in coral reefs of the Mexican Caribbean reported an average biomass density of 12 mg/cm2.52 We used this data to assign the coral agents a density of 0.0012 kg/m3 (assuming the samples in that study were 1 cm tall). In To adhere/grow corals section, we define the process of adhesion and growth of coral agents.\n\nGlobal variables are those common to the entire model, which all agents can access to carry out processes. In the Somers ABM, global variables include gravity, the salinity and density of the seawater, the direction and speed of the sea currents, the simulated month and year, and the salvage moments. Almost all of these variables are used by agents to compute different processes as described below.\n\nThe force of gravity in the ABM corresponds to the acceleration with which the objects will “fall” i.e., change their position with respect to the Z-axis. The value assigned to this variable is 9.81 m/s2, which corresponds to the gravitational pull at sea level. It should be clarified that the final falling speed of objects is not only a function of gravitational attraction but also of their mass, volume, and resistance given by water density so the speed must be calculated as part of the movement process.\n\nThe salinity and density of seawater are also defined as global variables. Various authors have reported salinity values for different points of the Veracruz Reef System, ranging from 33.6% to 38%.46,53–55 Based on these data, we assumed an average value for the global salinity variable of 35%.\n\nOn the other hand, seawater density is a function of both salinity and temperature. According to our own data obtained by scuba diving in different points of the Veracruz Reef System, including the Somers shipwreck, the water temperature below 20 m depth remains around 23 °C regardless of the season (dry or rainy). Considering this temperature and a salinity of 35%, the seawater-density in the ABM is 1024 kg/m3.\n\nThe current-direction and current-speed global variables are defined for the different months of the year based on the measurements made in Isla Verde by Salas-Pérez et al. Their measurements covered the entire water column at the sampling sites and are reported as vertical averages, therefore, we considered them adequate for the ABM in which the interactions will take place at the seabed level. Although the unit of distance at sea is the nautical mile (equivalent to 1852 metres or a minute of arc on the compass) and the speed unit is the knot (nautical mile/h), for the ABM we decided to keep units in km/h for consistency with those reported by Salas-Pérez et al. For the spring months (April-June) current direction flows towards the NW at 0.37 (± 0.40) km/h (mean ± s.d.). In summer (July-September) the direction is towards the SW at 0.24 (± 0.36) km/h. In autumn (October-December) the current goes to the NE at 0.26 (± 0.40) km/h. During the winter months (January-March) it flows to the SE at 0.84 (± 0.28) km/h.56\n\nThese values are not constants in the ABM. A random-normal function is used to produce normally distributed float random numbers, with the specified mean and standard deviation. In this way, each time the current direction and current speed variables are used, their values change within the range of the real measurements. For this reason, these two variables, unlike the rest of the global variables, are not assigned at the start of the simulation.\n\nThe current-month and current-year are also defined as global variables that determine, respectively, the month and year being simulated. Its initial values are “December” and “1846” corresponding to the date the Somers wrecked. How these variables are treated is specified later, in Time section.\n\nFinally, the global variable salvage-moments determines the moments of the simulation in which the Salvage sub-process is executed (see To salvage section). The values for this variable are stored in a list and are generated randomly with the maximum possible value being 2064, the number of months that make up the 172 years that passed from the Somers shipwreck until 2018 when the archaeological record of the site was carried out by PAMGI.\n\nThere are two major “moments” in the design and operation of an ABM: 1) “setup” when the user establishes the general parameters on which the simulation will be based i.e., the system’s initial conditions; and 2) “run” when the processes are executed. These two moments are designed and implemented in the two major processes of the ABM that we call Configuration and Execution. Figure 11 shows a flow chart of the ABM, including the processes and operations of both moments.\n\nThe first sub-process in the simulation’s setup defines the area that will represent the seabed and its attributes. The seabed is located at the bottom of the world, immediately adjacent to the ship's bottom. As mentioned previously, agents representing the seabed must create a random number of 1 to 10 sediment-class agents on each centimetre of their surface, which will allow simulating the movement of the bottom sediment and the formation of scour pits.\n\nThe pseudocode [1] for this sub-process is:\n\n• Locate seabed\n\n○ seabed Z-coordinate = false keel Z-coordinate\n\n• Ask seabed agents\n\n○ create between 1 and 10 sediments on every centimetre of their surface\n\nIn this sub-process, the 3D models that make up the parts of the ship are imported into the simulation environment. Although the geometry of the 3D models is made up of vertices and edges that form meshes of triangles, in the simulation each part of the ship constitutes an individual agent. Therefore, the behaviour of the agent must occur at the level of the complete mesh that makes it up and not at the level of the individual vertices or edges that make up the said mesh.\n\nImmediately after loading each piece, the class of agent to which it belongs is assigned, so that it already has the corresponding attributes. As mentioned before, the density and mass values of each agent depend on the material they represent. As part of this sub-process, density is first assigned to each agent and based on this and its volume, mass is calculated. Subsequently, initial values of each of the attributes that are specific to the agent are assigned.\n\nIn this step, the initial values of the different global variables are assigned. The pseudocode for this process is:\n\n• Establish\n\n○ gravity = 9.81\n\n○ salinity = 35\n\n○ seawater-density = 1024\n\n○ current-month = 12\n\n○ current-year = 1846\n\n○ current-direction = NW-->SE\n\n○ current-velocity = random-normal with mean = 0.84 and s.d = 0.28\n\n○ salvage-moments = choose n (n = total-salvages) between 1 and 2,076\n\nOnce the simulation’s initial conditions have been established in the Configuration process, we next define all those sub-processes that will imply actions and interactions between the agents. These sub-processes are included in the process called Execution.\n\nAs mentioned in Shipwreck site formation processes section, Muckelroy’s model considers three types of sub-processes: 1) those that remove elements from the system (extracting filters), including the shipwreck event, salvage operations, and the disintegration of perishable objects; 2) those that alter the spatial arrangement of objects (scrambling devices), including the shipwreck event and seabed movement; and 3) deposition of materials, including sediments and marine life. Conversely, Gibbs’ model considers the same general processes but makes them more specific and detailed, emphasising the decisions behind the actions. For example, in Muckelroy’s model, salvage operations constitute a single sub-process, but are subdivided in Gibbs’ model into crisis salvage, survivor salvage, systematic salvage, and opportunistic salvage.\n\nBased on the above, in the Execution process of the Somers ABM, the series of actions (sub-processes) that are executed at each simulation moment are: 1) to salvage, 2) to degrade, 3) to grow corals, and 4) to move (simulating both movement and sediment deposition). Since all processes involve actions in time, we first need to define time in the ABM.\n\nIn an ABM, the Execution process (with all its sub-processes) can be executed once or repeated cyclically. This last case is the most commonly used when modelling complex systems since it allows agents’ actions and interactions to be simulated iteratively over time. Thus, each iteration of Execution represents a unit of simulation time. Time units are arbitrarily defined according to our research questions and can represent real units (seconds, hours, years) or be dimensionless.\n\nTo determine the amount of time each Execution cycle would represent in the Somers ABM, we considered the units used to report the data we used for defining the different sub-processes. Thus, data about the movement of marine currents were reported by the seasons of the year i.e., quarterly,56 while studies on wood and metal degradation, as well as on the growth of colonising organisms, reported data at intervals of six months or one year.50,57,58 Based on this, we decided that the unit of time that would represent each moment of Execution in the ABM would be one month.\n\nFrom this definition of time, we needed to adjust the mentioned data to said time unit. In the case of the sea current during the summer months, for example, it was reported to flow in a SW direction at an average speed of 0.24 (± 0.36) km/h.56 Therefore, it was necessary to convert these current velocity data to metres per month.\n\nA problem that can arise when using units of time such as a month is that the movements of the elements in a single iteration of the ABM can be very large. For example, 0.24 km/h would imply a displacement of 173 km in a month. To avoid this problem, we programmed the ABM so that the salvage, degradation, and organism growth sub-processes (see below) stop whenever something is moving, and resume when movement stops. The latter is calculated as a differential between the position of every element at the beginning and the end of the iteration.\n\nFrom the above, it follows that the ABM should be able to keep a record of the month to which the Execution cycle that is running at a given moment corresponds. The global variable current-month is used for this purpose, which has values from 1 (January) to 12 (December), and in each iteration of Execution increases by one unit (returning to 1 after 12). Additionally, the global variable current-year keeps track of the year being simulated and increases one unit each time the month changes back to “1” (January). The initial values of current-month and current-year are “12” (December) and “1846”, respectively, corresponding to the date the Somers sank.\n\nFurthermore, to be able to analyse the SFP after a specific time has elapsed, the variables end-month and end-year are used to define the date on which the simulation should end. The default options are defined for August and 2018 so that the simulation covers the time passed from the shipwreck until the photogrammetric record of the wreck was carried out by PAMGI59; however, the user can modify both values. The simulation stops when it reaches the dates set in these parameters.\n\nThe pseudocode for time-related actions is:\n\nBefore simulation starts (SETUP):\n\n• set current-month = 12\n\n• set current-year = 1846\n\nOn every Execution:\n\n• if current-month < 12, set current-month = current-month + 1\n\n• if current-month = 12, set current-month = 1\n\n• if current-month = 1, set current-year = current year + 1\n\n• if current-month = end-month AND current-year = end-year, stop the simulation\n\nThis sub-process simulates both contemporary and post-wreck salvage operations. In Muckelroy’s model and even more so in Gibbs’ model, salvage events are central to shipwreck SFP as they have the potential to significantly alter the distribution of objects observable in the archaeological context. That is, to generate great differences between the systemic context and the archaeological context. In Muckelroy’s model, all these actions are grouped into the salvage operations sub-process. In Gibbs’ model, contemporary salvage operations fall into the first three categories of crisis salvage, survivor salvage, and systematic salvage; while subsequent operations fall into the fourth category, opportunistic salvage.\n\nUnlike the previous processes in the ABM where user input is minimal, this sub-process relies heavily on the information the user enters before starting the simulation. This information necessarily comes from historical research about the number of salvage operations that have been carried out on the wreck, from its sinking to the present day. Of the four types of salvage operations proposed by Gibbs, at least the first three should leave some kind of historical record, which may be as diverse as survivors' accounts, ship's logs, official documents issued by those responsible for the ship or the fleet (captains, commodore), newspaper notes, paintings, literary works, or photographs. The fourth type of operation, opportunistic salvage, is much less likely to leave a historical record as these operations are often the result of illegal salvage, the recovery of objects by local inhabitants, or recreational divers. However, it is possible to have data on this type of operation, which is why they are also considered in the ABM.\n\nFor the simulation to take into account the four types of salvage operations, we included selectors for each of them in the user interface. These are:\n\n• number-of-salvage-operations-during-the-crisis\n\n• number-of-salvage-operations-by-survivors\n\n• number-of-systematic-salvage-operations\n\n• number-of-opportunistic-salvage-operations\n\nIf the user knows these numbers from historical data, the number of corresponding salvage operations can be determined in each selector, with options ranging from 1 to 10. Additionally, we included a random number option (limits 0 and 10) in each selector to be able to simulate different scenarios when the exact number of salvage operations is unknown. In the case of the Somers, for example, the documents we have consulted so far do not mention any salvage operation being carried out contemporary to the sinking, so the initial values of the first three selectors would be zero.\n\nBeing able to distinguish between types of salvages allows archaeologists to have greater precision in the story we tell about the SFP since both the motivations behind the salvages and the potential number of objects removed will be different in each salvage type. However, for simulation purposes we considered all salvage operations to be equivalent in terms of the number of objects removed from the environment as a result of each operation. In other words, in the ABM, as many objects can be lost from a systematic salvage operation as from an opportunistic one. This assumption is not necessarily true in historical terms, but we made it so for the sake of simplifying the ABM.\n\nTherefore, for this sub-process, the total number of salvage operations carried out on the wreck from its sinking to the present is of interest. This number is stored in a global variable named total-salvages, the result of adding the values of the four selectors described above. It is defined after the initial setup of the environment but before the start of the simulation.\n\nSince each time step of the ABM corresponds to a month, the Salvage sub-process should not be executed in each iteration because it would imply that salvage operations would have been carried out every month since the sinking, which would be a mistake. Instead, the sub-process should only be executed at certain moments, whose total number would be defined by the total-salvages variable, throughout the simulation. To determine at what times the sub-process runs, a list of all possible moments is created, and the number of times is selected randomly.\n\nGiven that, according to Gibbs’ model, crisis salvage operations, survivor salvage, and systematic salvage are contemporaneous with the shipwreck event, the salvage moments corresponding to these three variables occur in the first 24 months. Consequently, this simulates that these operations would have taken place between the years 1847 and 1848, that is, during the Mexican-American War. In contrast, the moments corresponding to opportunistic salvage can occur in any month from January 1849 to the end date of the simulation, with a maximum value of 2,064, the number of months in the 172 years elapsed from the sinking of the Somers to PAMGI’s recording. The values of the months in which salvage operations are to be simulated are stored in another global variable called salvage-moments.\n\nThe salvage-moments variable is calculated immediately after total-salvages so that its values are already set in the global variable when the simulation starts. The execution of the Salvage sub-process occurs when current-month is equal to one of the numbers in the salvage-moments list.\n\nTherefore, to simulate the loss of objects due to salvage operations, when the Salvage subprocess is executed, a probability of disappearing from the environment is calculated for each piece of the ship, which is directly proportional to its salvage-value (SV) and inversely proportional to its salvage-difficulty (SD). The result of dividing these two values (SV/SD) is normalised by dividing by the maximum value of SV/SD of all the agents so that it constitutes a probability value, i.e., a positive number between 0 and 1. For each piece, this number is stored in a local variable called the disappearance-probability. Thus:\n\nIn the case of the Somers, something that would undoubtedly have challenged salvage operations in the months or years following its sinking and until the middle of the 20th century, was the depth at which the wreck is located (32 m). But that difficulty has lessened considerably since the mid-1940s with the invention of scuba diving equipment by Cousteau and Gagnan in 1942. To take this into account, when the current-year value reaches 1950, the salvage-difficulty value of all agents is reduced by 50%. This percentage is arbitrary but can be changed at any time.\n\nFinally, to increase stochasticity and simulate in a certain way the possible success or failure of the rescue operations, if an agent’s disappearance-probability is greater than 0.5, a new local variable named disappearance-success with a random value between 0 and 1 is calculated. If disappearance-probability is greater than disappearance-success, then the piece disappears from the environment. This implies that, even when the simulation of a salvage operation is programmed and the sub-process executed, it does not necessarily result in an agent’s disappearance, since the latter is a function of said disappearance-success.\n\nThe pseudocode for this sub-process is:\n\nBefore simulation starts (during Configuration):\n\n• set the number of salvage operations on each selector\n\n○ initial values: first three = 0, opportunistic salvage = random up to 10\n\n• calculate total-salvages = sum of four selectors\n\n• calculate salvage-moments\n\n○ random up to 2,064\n\nOn every Execution:\n\n• if current month = any of the items in the salvage-moments list, execute Salvage\n\n• for each agent\n\n○ if current year ≥ 1950, reduce salvage difficulty by 50%\n\n○ calculate disappearance-probability = (SV/SD) / max SV/SD\n\n○ if disappearance-probability > 0.5\n\n■ compute local variable disappearance-success → random between 0 and 1\n\n■ if disappearance-probability > disappearance-success, remove the piece from the environment.\n\nThis sub-process simulates the degradation of wooden elements and corrosion of metallic elements. To make it easier to verify the correct operation of this process, we divided it into two sub-processes: degrade-wood and degrade-metal.\n\nDegrade-wood\n\nWood degradation in archaeological contexts, both terrestrial and submerged, depends on several factors including the type of wood and the time it has been in the context, as well as its particular characteristics such as moisture content, oxygen, pH, temperature, and, in marine contexts, salinity. While in most archaeological contexts microorganisms (bacteria and fungi) are the main agents responsible for wood degradation,60,61 in marine contexts with warm water conditions and abundant light, wood is rapidly degraded by boring animals long before degradation by microorganisms begins. Bivalve molluscs of the Terenidae family, especially Teredo navalis, and crustaceans of the genus Limnoria are the main organisms responsible for drilling exposed wood. The specific prevalence of the different species of borers varies significantly depending on environmental conditions, particularly temperature and salinity.62 However, both in warm (16–26 °C) and cold waters (5–12 °C), these animals can completely degrade a piece of wood between six months and a year, depending on the size and type of wood.57,58\n\nDegradation by boring animals is limited to the exposed parts of the submerged wood and is completely absent in those parts covered by more than 10 cm of sediment. In these cases, degradation does continue, although much more slowly, due to the action of soft rot fungi, tunnelling bacteria, and erosion bacteria. Below 40 cm of sediment, conditions become almost anaerobic and the wood degradation process is drastically reduced, being carried out exclusively by erosion bacteria that can proliferate in such conditions.58 Therefore, the degradation of wood-representing agents in the ABM depends on whether they are exposed or covered by sediment.\n\nBased on experimental data reported by Pournou et al., and Björdal & Nilsson, we developed the following linear regression equations for estimating the monthly degradation of oak and pine woods, both exposed and covered by sediment.57,58 A detailed explanation of how we developed these equations can be found in Ref. 70.\n\nThese calculations correspond to the Execution phase of the ABM. However, as mentioned previously, before the simulation starts wood agents have a degradation-percentage randomly set between 0 and 10% to add stochasticity to the model by simulating that pieces did not have complete integrity at the time of the shipwreck.\n\nIt was common practice to cover the hulls of wooden ships with metal sheets to reduce wood degradation since the corrosion products of said metals are toxic to boring organisms. Thus, the metallic sheathing conferred protection against degradation to those parts of the ship that would be permanently submerged, i.e., the bottom of the hull and the rudder blade. To simulate such protection in the ABM, in wooden elements that are in contact with brass parts, the degradation-percentage is set to only 10% of the result of the normal degradation calculation. This percentage of “protection” was arbitrarily set but can be modified at any time based on experimental data.\n\nThe pseudocode for the degrade-wood sub-process is:\n\nBefore simulation starts (during Configuration):\n\n• set degradation-percentage = random between 0 and 10\n\nOn every Execution:\n\nFor all agents with wood:\n\n• calculate cm-sediment = sum of the sizes of sediments and corals adjacent to the agent × 100\n\nFor wood-oak agents:\n\n• calculate sediment_percentage=cm_sediment/100\n\n○ if sediment_percentage > 0, calculate degradation_percentage=4.52×x−5×1−sediment_percentage)+−0.00056×z+0.02831000agent_size×sediment_percentage\n\n■ if adjacent to brass, set degradation_percentage=degradation_percentage×0.1\n\nFor wood-pine agents,\n\n• calculate sediment_percentage=cm_sediment/100\n\n• if sediment_percentage > 0, calculate\n\n○ degradation_percentage=8.33×x−5×1−sediment_percentage)+−0.000928×z+0.05471000agent_size×sediment_percentage\n\n■ if adjacent brass, set degradation_percentage=degradation_percentage×0.1\n\nDegrade-metal\n\nThe degradation of metallic objects in submerged archaeological contexts results from their interaction with the environment and colonising organisms. The former produces corrosion as a result of an electrochemical reaction that depends on the metal’s electrode potential and the medium’s pH.63 On the other hand, the development of colonising organisms and their concretions exert a protective effect against corrosion because, with their growth, they limit the exposure of the metal to the environment.\n\nAs we did for wood degradation, for metal elements we also developed linear regression equations for estimating the monthly corrosion of iron and brass pieces based on experimental data reported by López Garrido et al.50 A detailed explanation of the development of the following equations can be found in Ref. 70.\n\nCorrosion of metal agents is visually represented in the simulation with a change in colour towards dark red or green depending on whether the agent represents iron or brass, respectively. As aforementioned, before starting the simulation, metals have a corrosion-percentage randomly set between 0 and 5%, simulating that the pieces did not have complete integrity at the time of the shipwreck.\n\nThe pseudocode for this sub-process is:\n\nBefore simulation starts (during Configuration):\n\nFor all metal agents:\n\n• set corrosion_percentage = random between 0 and 5\n\nOn every Execution:\n\nFor metal-iron agents:\n\n• calculate\n\n○ if corrosion_percentage < 0, set it to 0.\n\n• if corrosion_percentage > 50, set colour to dark red\n\nFor metal-brass agents:\n\n• calculate corrosion_percentage=0.2×elapsed_months+0.133\n\n○ if corrosion_percentage < 0, set it to 0.\n\n• if corrosion_percentage > 50, set colour to green\n\nDegrade-rigging: the great missing\n\nAlthough a ship’s structure and equipment (e.g., artillery, anchors) usually occupy much of the attention during the study of a wreck, a less attractive but equally important component is all the elements that make it possible for the ship to move. These are rigging and sails.\n\nRigging is “the whole of all the cordage of a ship, and the title of every whole piece of rope”.42 In addition to the ropes, it is composed of several very diverse elements that facilitate their movement, collectively called blocks (Figure 12). Although rigging usually receives many different names depending on its location or function on the ship, we can generally speak of two types: standing rigging, the one that does not move and whose function is to provide “fastening for masts and topmasts”; and running rigging, the one that “is in play [i.e., in motion] for the handling of the entire rig”42 (Figure 13).\n\n(Adapted from Ref. 72).\n\nAs highlighted by Sanders in his detailed article on the characteristics of ropes and their archaeological record, a ship's rigging was not simply an accessory but\n\n“[...] a major part of the investment in a ship, requiring major industry to support it. [...] A French frigate of 1790 required 27 km of cordage for rigging and a further 5 km for replacements. [...] A late-18th-century British 74-gun ship required c.80 tons of rope to rig it, and some 922 blocks”.64\n\nAlthough cordage and sails of ships before the 20th century were made of perishable organic materials, it is not uncommon to find remains of these elements in archaeological excavations of shipwrecks. They are often adhered to pieces of iron or covered with calcareous concretions, as complete pieces impregnated with tar or a similar compound, or as complete or fragmented pieces deposited in the sediment and very prone to disintegrate during excavation.65\n\nTherefore, models of shipwreck SFP should include at least some rigging elements. However, we decided not to include these elements nor their degradation process in the Somers ABM for two reasons. Firstly, the line plans published by Chapelle only show the dimensions of the structure, masts, and sails (see Figure 4), but do not give details about rigging. This does not constitute an omission on Chapelle’s part, much less the original author of the plans (the shipbuilder). It is simply that the rigging, both standing and running, did not require a specific plan since its quantity and dimensions were adapted according to the dimensions of the masts and sails specified in the construction plans.\n\nAn alternative for adding rigging to the 3D model of the Somers would be to rely on Nathaniel Currier's lithograph of the ship (Figure 3). However, it must be considered that the rigging shown in a ship’s representation, whether pictorial, sculptural, or a 3D model, does not necessarily correspond to the totality of the rigging carried in the ship. This is due to several reasons. Firstly, “artists were seldom riggers or shipwrights, so pictures are seldom accurate. Rigging may obscure the composition and is not always aesthetically pleasing, so is only selectively portrayed. Models seldom have their original rigging, and problems of scale mean that the detail of the real object cannot be replicated. A further limitation of rigging on models is that they are representational or instructional”.64\n\nThis would imply that even based on Currier’s lithograph or any other representation of a ship of the time, the 3D model of the Somers would not have all the elements of the rigging and its accessories. Although this constitutes a limitation, it is an inherent characteristic of all models, in that they represent only those aspects of reality that allow answering specific questions about it.\n\nThe second reason for not including rigging and its degradation process in the Somers ABM is that we do not have experimental data about it. The degradation of textiles in humid archaeological contexts occurs mainly by bacterial action, either mechanical (by penetration and growth within the fibres) or chemical (by enzymatic hydrolysis).66 In most of the studies that have analysed such degradation, textiles have been buried in various types of soils with different moisture conditions.66 However, such studies have included different types of fabrics, but no ropes. As far as we know, there are no experimental studies where the rigging degradation processes have been specifically analysed, similar to those on which we relied for designing the degradation processes of wood and metals. This would be an interesting and necessary research topic to delve into the knowledge of shipwreck SFP.\n\nThe process of biological colonisation of submerged materials is one of great complexity and specificity between organisms, be they bacteria, algae spores, or larvae. However, in all cases it involves four main processes: 1) transport to the surface of the substrate, 2) settlement, 3) attachment, and 4) growth. Since the biological colonisation process was not this work’s subject, for the Somers ABM we simulated the first three stages (transport, settlement, and attachment) as a single “adherence” event, which was defined to be random. That is, we assigned a random probability for colonising organisms (i.e., coral agents) to appear on the surface of the agents that represent metals. In this way, for the simulation, we concentrated only on specifying the growth conditions of these organisms.\n\nIn the ABM, the biological colonisation process is limited to agents that represent metals and is differential between iron and brass. We based growth rates for both metals on experimental data reported by two studies carried out in the Gulf of Mexico.50,67 The detailed rationale for determining such growth conditions can be found in Ref. 70.\n\nFor elements representing iron, we defined that biological colonisation would advance at 1.547 cm per month, while the rate of growth on brass pieces is 2.3% of their size per month.\n\nThe pseudocode for this process is:\n\nTo simulate adherence of colonising organisms:\n\nFor all metal agents\n\n• if they don't have coral on top\n\n○ “toss a coin” → if random-float (range 0-1) > 0.8, sprout a coral\n\n(note: this 20% probability of appearing was set this way to avoid performance problems in the simulation since it might quickly be saturated with coral agents)\n\nTo simulate the growth of colonising organisms:\n\nFor corals in iron\n\n• if size < size of the metal-iron agent on which it is\n\n○ set size=size+0.01547\n\nFor corals in brass\n\n• if size < size of the metal-brass agent it is on\n\n○ set size=size+size∗0.023\n\nIn terms of Muckelroy and Gibbs’ SFP models, agent movement allows to simulate:\n\n1. Object loss by flotation. In Muckelroy’s model, this corresponds to material floated away as a result of the process of wrecking (Figure 1). In Gibbs’ model, this type of removal action is found in the pre-impact warning and impact phases (Figure 2).\n\n2. Object loss due to jettisoning. While this type of action is not considered in Muckelroy’s model, in Gibbs’ model it occurs during the pre-impact warning and impact phases (Figure 2).\n\n3. Object deposition by gravity and sedimentation. In both Muckelroy and Gibbs’ models, material subsequently deposited on site occurs after the sinking process, as one of the scrambling devices, constitutes the natural events of site formation (Figure 1 and Figure 2).\n\nIdeally, the simulation platform to be used should have a physics module that allows adequately simulating agents’ movement (e.g., Unreal Engine). Having this functionality greatly simplifies the programming and performance of the ABM. However, in case the platform does not have a physical movement simulation module (e.g., NetLogo), in Supplementary material 370 we provide an alternative for encoding the movement process of mobile agents. For each agent this calculates: 1) the displacement velocities in each of the three dimensions (X, Y, and Z) as a function of current velocities and seawater density, and 2) the new position of the agent after applying said displacement velocities.\n\nTo be able to test historical and archaeological hypotheses we need to generate a set of indicators that would allow comparing the results of the simulations to data obtained from historical documents and the archaeological record of the wreck. As mentioned earlier, this corresponds to a model validation of the empirical macro validation type.\n\nNext, we present some basic indicators considered necessary for testing hypotheses. These should be visible in the user interface throughout the simulation and, most importantly, they had to be exported to a text file or similar for later analysis.\n\nWe added an indicator called Simulated date to know what month and year the current moment of the simulation corresponds to, in addition to being used in other indicators. This indicator simply combines the global variables current-month and current-year.\n\nThe distribution of wreck elements on the seabed is the main indicator for considering the ABM validated. It is also the main output or outcome variable of the shipwreck SFP considered in both Muckelroy and Gibbs’ models (observed seabed distribution).\n\nIn terms of representation, the distributions of elements in an archaeological record can be displayed in different ways, depending on the detail required for analysis. One way is to represent them as forms, that is, presenting their contours or volumes. This is what archaeologists usually do when drawing a context in a plan or section. It is a relatively undetailed representation but undoubtedly useful for recording and interpreting contexts. Another much more detailed method is to represent the context as a continuous array of points in three-dimensional space. This type of representation is called a point cloud and is the kind that results from a topographical survey or a photogrammetric record, for example.\n\nIn the case of the Somers wreck, from the photogrammetric record carried out by PAMGI in 2018, a very dense point cloud was obtained that represents the distribution of wreck elements in great detail (Figure 14). In the ABM, to compare the results of the simulations with said photogrammetric record, it was necessary to obtain a point cloud from the simulation. Points are given by each of the vertices that make up the 3D meshes of the elements of the wreck. These points are obtained by exporting the data of the coordinates of all vertices (i.e., their positions in X, Y, and Z) of all remaining agents at the end of the simulation to a comma-delimited text file.\n\nAbove: a model of the complete wreck. Below: a detail of the bow area.\n\nFrom Ref. 7 Reproduced with permission from Aspha Ediciones. Both models by Rodrigo Pacheco y Felix Pedrotti.\n\nThe pseudocode for this export process is:\n\n• create CSV file in a user-defined location\n\n• from each remaining agent’s mesh, obtain each vertex ID and position\n\n○ write ID, X, Y, and Z of vertices in the text file\n\nFrom the above it follows that we should then be able to compare two point clouds, the one obtained from the simulation against the one from the archaeological record. For this, a number of open-source tools are available (e.g., CloudCompare) that would yield the differences between point clouds both visually and numerically by calculating a correlation value between them. Based on these data it would be possible to 1) decide whether the ABM is valid or not based on said correlation value and its statistical significance, and 2) make interpretations about the SFP by analysing the conditions of those simulations that result in higher correlation values.\n\nThe foregoing, however, assumes that the point clouds obtained from the simulations will have a sufficient level of detail to be able to compare them with the photogrammetric record. However, if for any reason it is not possible to compare the distributions of wreck elements at the “microscopic” level of detail of the point clouds, then it should be done at the “macroscopic” level, from the analysis of objects and their relative positions.\n\n\n\nThis indicator allows exploring how much of the ship’s structure was lost during the shipwreck. It reports the difference between the number of structural elements of the ship, both major and minor, at the beginning of the simulation and at the end of the first month.\n\nThe pseudocode for this reporting process is:\n\n• report (sum of major structural elements at the end of month 1 of simulation / sum of initial major structural elements) × 100\n\n• report (sum of minor structural elements at the end of month 1 of simulation / sum of initial minor structural elements) × 100\n\nIn terms of the historical explanation of the SFP, another necessary indicator is the number of salvage operations carried out on the shipwreck, both contemporary (systematic) and after the sinking (opportunistic). To the user interface and the text file of the simulation results we added an indicator of the total number of salvage operations. As mentioned in To salvage section, this number is stored during the configuration step in a global variable called total-salvages, which is the result of adding the values of the four types of salvages. Additionally, for systematic salvage operations, a similar indicator is used which limits the results to the period of the Mexican-American War (December 1846–February 1848).\n\nThe pseudocode for this reporting process is:\n\n• report total-salvages\n\n• report salvage count if current-year = 1847 or 1848\n\nThis indicator allows exploring how quickly the shipwreck would be covered by sediment deposition. It can be used to test, for example, various sea current conditions or sediment concentrations. For this indicator, we programmed a reporting process in which, at the beginning of each year, the area of the wreck that is above the maximum height covered by the sediment deposits is calculated.\n\nThe pseudocode is:\n\n• if current-month = January\n\n○ calculate wreck’s original-height = Zmax_original − Zmin_original\n\n○ obtain maximum sediment height (Zsediments)\n\n○ calculate wreck’s current-height = Zmax_current − Zsediments\n\n○ calculate covered wreck percentage=1−current_height/original_height×100\n\nThis indicator allows the exploration of how much of the mobile elements located on the wreck’s surface was lost as a result of opportunistic salvage operations (i.e., those after the end of the Mexican-American War). Therefore, the process that generates the indicator only considers events after February 1848.\n\nThis indicator is linked to the Salvage process so that, if the latter is successful and the current-month variable corresponds to an opportunistic salvage moment, a report is generated with the name of the lost element and the corresponding month.\n\nThe pseudocode is:\n\n• if (simulated-date ≥ February 1848)\n\n○ if (disappearance-success = true) AND (current-month = salvage-moment), report “missing item name, current-month”\n\nThis indicator allows the exploration of how much of the ship's structure has been lost as a result of wood degradation. It reports the percentage of structural elements, both major and minor, lost at the end of the simulation.\n\nThe pseudocode for this process is:\n\n• at simulation end\n\n○ report (final structural element count / initial structural element count) × 100\n\nFrom our point of view, the central objective of studying archaeological sites from the perspective of SFP is to be able to answer, in as much detail as possible, the question: what happened here? That is, we seek to tell a story. Since telling a story necessarily implies the passage of time, it is essential that we can sequentially locate the events, whether natural or social, identified in the SFP. Obviously, the more detailed the chronological sequence, the more detailed the story we could tell.\n\nObtaining coherent chronological sequences of natural and social events is the goal of archaeological stratigraphy. However, since the stratigraphic analysis of a site is naturally linked to its stratigraphic excavation, establishing such a sequence will be almost impossible if the site is not to be excavated. In the best of cases, we would only be able to tell part of the site’s history, that of the systemic context, if the historical sources allow it. However, we would hardly be able to tell the second part of the story, that of the archaeological context.\n\nThis is where SFP simulation acquires great relevance as an interpretive tool since it allows us to recreate historical events to a certain extent. Furthermore, by using computational platforms to carry out the simulations, it is possible to obtain a chronological record of absolutely all the actors, variables, and processes, with the required detail, even in real-time. For this reason, in the Somers ABM, we considered it essential to include a chronological record of two types of events: 1) degradation and 2) deposition of ship’s elements.\n\nA classical stratigraphic analysis would focus primarily on the sequence of element deposition.68 However, this assumes that the objects will degrade slowly enough that the archaeologist can observe them years or centuries after their deposition. This could be true for some submerged contexts whose environmental conditions are suitable for the conservation of archaeological materials, particularly wood (e.g., cold, deep water, with silt/clay sediments and anoxic environments). However, this is not usually the case in contexts located in tropical reef waters, such as the Somers, where environmental conditions favour wood degradation. In these contexts, an element may completely degrade in situ without ever being deposited on an interface (surface); such would be the case, for example, of the upper segments of the frames or hull planking. It may also be the case that objects fall and are deposited in the sediment but completely degrade after their deposition. In both cases, it would be very unlikely that archaeologists would find remains of such elements for them to be included in the stratigraphic sequence.\n\nTherefore, in the ABM we included a record of both degradation and deposition. The model records each time a wooden element reaches 100% degradation and each time an element, be it wood or metal, makes contact with a sediment surface. In both cases, the moment (in milliseconds of simulation) in which the event occurred and the months that have elapsed are recorded.\n\nThe pseudocode for these processes is:\n\nIn Configuration:\n\n• create “Degradation” and “Deposition” lists\n\nOn every Execution:\n\n• for each wooden element\n\n○ if degradation-percentage = 100, add to “Degradation”: element name + time (msec)\n\n• for all elements\n\n○ if it hits sediment bottom, add to “Deposition”: element name + seabottom name + moment (msec)\n\nHere we detail the components contained in the ABM’s user interface, although we have already referred to most of them in other sections. The user interface is made up of three elements: 1) setup controls, 2) process monitors, and 3) simulation results.\n\nThe different controls described here correspond to the various sub-processes considered in Gibbs’ model, constituting variables whose values are used in different processes. Since their values alter both the way in which the ship’s 3D model is presented and the ABM’s processes, these controls should be defined by the user before starting the simulation (Configuration process).\n\nIn terms of CAS, defining these controls establishes the system’s initial conditions, while their different permutations allow evaluating different properties of the SFP as a complex system, including possible tipping points and emergent properties. In this case, different configurations of the element distribution in the shipwreck.\n\nAlthough the definition of these controls can be completely arbitrary, ideally it should be based on historical data about the ship and its sinking process. Such was the objective of consulting historical sources about the Somers, presented in Historical sources on which the ABM is based section. Defining the initial conditions of the process in this way adds the dimension of human decisions to the SFP; that is, not only considering how the process happened but also why it happened that way.\n\nWe based these setup controls on Gibbs’ model, as detailed below.\n\n• Pre-impact — threat phase.\n\nThis sub-process refers to actions carried out, in the short and long-term, to avoid a naval accident. Long-term actions normally take place long before sailing, including the design of the ship itself, structural modifications, equipment, route, and even crew selection. Short-term actions are those taken during navigation when a threat may be perceived even though an imminent danger is not yet present.13\n\nIn the specific case of the Somers, the clearest long-term action was the metallic sheathing of the hull which, although not included in the ship’s plans, is present in the wreck. As for short-term actions, several are mentioned in Lt. Semmes’ and surgeon Wright’s accounts of the shipwreck, particularly after noticing signs of an approaching norther (i.e., strong wind or storm coming from the north), including increased alertness of captain and crew, manoeuvres to move away from the reef, and attempts to return to the anchorage site.28,29\n\nHowever, although these actions add a certain historical aspects to the explanation of the SFP, most of them cannot be included in an ABM. It would be necessary to resort to other simulations to test the effect of these actions on the sinking process. The only action that it is possible to include in the ABM is the metal sheathing that covers the bottom of the hull and rudder blade, which is included in the 3D model and loaded into the ABM as a metallic agent.\n\nConsidering the above, the user interface does not include controls associated with this part of Gibbs’ model.\n\n• Pre-impact warning phase\n\n○ Were anchors dropped?\n\n■ Controller options:\n\n• yes → anchors are removed from the 3D model\n\n• no (default) → anchors stay in place in the 3D model\n\n• Impact — crisis salvage phase\n\n○ The use of anchors can also be considered at this stage. However, since it was added in the previous one, it is not included again here.\n\n○ Were heavy objects jettisoned?\n\n■ Controller options:\n\n• no (default) → all elements of the 3D model stay\n\n• some → 0-50% (random) of carronades and cargo disappears\n\n• all → all carronades and cargo disappear\n\n○ Were masts cut? How many?\n\n■ Controller options:\n\n• 0 (default) → masts remain unchanged\n\n• 1 → on either mast, middle and top parts are removed\n\n• 2 → on both masts, middle and top parts are removed\n\n○ Were holes made on the hull?\n\n■ Controller options:\n\n• no (default) → 3D model remains the same\n\n• yes, repaired → 3D model remains the same\n\n• yes, without repair → a random number of holes, between 1 and 10, are created in the hull (removing components from the outer and inner planking)\n\n○ Were boats dropped?\n\n■ Controller options:\n\n• yes (default) → boat disappears from the 3D model\n\n• no → 3D model remains the same\n\n○ Were materials removed for survival?\n\n■ Controller options:\n\n• no (default) → 3D model remains the same\n\n• yes, accessories → a random number < 20% of total accessory items (element-category = 2) disappear\n\n• yes, minor structural elements → a random number < 20% of total minor structural elements (element-category = 3) disappear\n\n• yes, major structural elements→ a random number < 20% of total major structural elements (element-category = 4) disappear\n\n○ Number of crisis salvage operations\n\n■ Range 0 (default) to 10 (integers)\n\n• Recoil — survivor salvage phase\n\n○ Number of survivor salvage operations\n\n■ Range 0 (default) to 10 (integers)\n\n• Rescue/post-disaster phase\n\n○ Number of systematic salvage operations\n\n■ Range 0 (default) to 10 (integers)\n\n○ Number of opportunistic salvage operations\n\n■ Range 0 (default) to 10 (integers)\n\nAn important variable to consider in the analysis of the shipwreck SFP is the list of the ship, that is, if at the time of the shipwreck it leaned towards one of its rails and how pronounced this leaning was. This variable is not contemplated in Gibbs’ model but would affect the distribution of observable elements in the shipwreck.\n\nThe accounts of the Somers’ wreck mention it listed to starboard and thus went to the bottom. Since we don’t know precisely how many degrees it heeled over, we added two controls to the simulation’s configuration:\n\n• Did the ship list?\n\n○ Controller options:\n\n■ no\n\n■ yes, to port\n\n■ yes, to starboard (default)\n\n• Degrees of list\n\n○ Range 0 to 90°. Note: we determined this range considering the specific case of the Somers. However, since it is possible that a ship’s hull may turn 180° during a shipwreck, this range should be extended to 360° in future versions of the ABM to give more freedom to this parameter.\n\nFinally, we added End simulation month and End simulation year selectors to allow the user to enter a specific date for ending the simulation and explore what the conditions of the shipwreck and the different indicators would have been on that date. The default options are defined as “August” and “2018”, the date on which the photogrammetric record of Somers was carried out by PAMGI.59\n\nThe second component of the user interface is monitors, non-user-modifiable indicators that are visible at all times throughout the simulation, providing relevant information in real-time. Monitors include:\n\n• Simulated date: shows which month and year the current moment of the simulation corresponds to.\n\n• Elapsed months: the number of “months” that have passed since the start of the simulation.\n\n• Salvages: shows the number of salvage operations programmed in the configuration, both total operations and each of the four types of salvage.\n\n• Wood degradation percentage: the global degradation percentage of all remaining wood agents in the simulation.\n\n• Metal corrosion percentage: the overall corrosion percentage of all remaining metal agents in the simulation.\n\n• Number of remaining pieces: the total number of pieces left in the simulated shipwreck.\n\n• Covered wreck percentage: the proportion of the remaining volume of the wreck that is covered by sediment agents.\n\n• Simulation duration: the elapsed time, in minutes and seconds, since the simulation started.\n\nThe last element in the user interface is a dialogue box showing the results of the simulation, i.e., the indicators mentioned in the previous section. It includes:\n\n• end date of the simulation\n\n• percentage of structural elements lost, both total and due to shipwreck\n\n• number of salvage operations (total and of each type)\n\n• number of mobile elements lost due to shipwreck\n\n• number of pieces remaining\n\nAdditionally, the dialogue box provides the user with an option to export the simulation results. If chosen, the process of creating text files for both results and point cloud and exporting them to a user-defined location is executed.\n\nTo explore the Somers shipwreck SFP and test different historical hypotheses, the ABM should start from a set of initial conditions, resulting in quantifiable differences in the different indicators, particularly the distribution of elements in the wreck. Such conditions are based on historical data of the wreck, so for those where historical records are available, conditions remain constant. Meanwhile those for which there is no historical evidence are variables that can be explored in the simulation. Thus, for example, the accounts of the wreck of the Somers do not mention that the anchors were dropped but they do mention that the boat was launched; there is also no mention of crisis or systematic salvage operations. These data should remain constant in all simulation scenarios. On the contrary, since there are no records on opportunistic salvage operations, this remains a variable from which different scenarios can be simulated.\n\nBased on our review of historical documents related to the Somers wreck (Historical sources on which the ABM is based section), the initial conditions that should remain constant for its SFP simulation are:\n\n• anchors were not dropped\n\n• heavy objects were not intentionally jettisoned\n\n• masts were not cut\n\n• no holes were made in the hull (since the ship did not crash)\n\n• the boat was launched\n\n• there was no intentional removal of survival materials\n\n• there were no crisis salvage operations\n\n• there were no survivor salvage operations\n\n• there were no systematic salvage operations\n\n\n4. Discussion\n\nIn 2008, Mexican maritime archaeologist Jorge M. Herrera wrote in his doctoral thesis about the possibility of experimentation in archaeology: “Maritime archaeology is sadly limited in its options in regards to experimentation, as it would be rather impractical to go [...] and wreck several boats to see if the conceptual models matched reality”.19\n\nThis statement is directly related to the nature of archaeologists’ object of study since we face the challenge of trying to narrate an entire film by seeing only the last scene. We put together conceptual models, hypotheses, and interpretations about what could have happened during the film in order to end up with this scene. But the scene we see, the archaeological context, is not a fossil frozen in time but the result of interactions between many factors over many years.8 Furthermore, as the magnitude and type of such interactions have surely changed over the years, the site formation process is not linear, which leaves us with a wide range of possible interpretations of stories to tell. How, then, can we distinguish among the range of possible stories which one(s) are most likely to have occurred and be best suited to telling the site’s story? The task of discerning the SFP is even more complicated when we consider that archaeologists are working with an “unrepeatable experiment”,69 especially when it comes to excavation. That is, we cannot repeat history to prove what could have happened and what could not. It is not practical, as Herrera said, to sink several boats to see which conceptual model most closely resembles reality. We can't rebuild the USS Somers and sink it again … can we?\n\nToday, we are less limited in our capacity for experimentation in maritime archaeology. Through the use of computational tools such as ABM, we can make the experiment repeatable. We can test different conceptual models of the SFP, simulate different scenarios with different process conditions, and see in what archaeological context they result. We can put together and dismantle the Somers as many times as we want. Such is our proposal: that from the standpoint of CAS, an ABM could serve as a tool for postulating a sustained interpretation of a shipwreck’s SFP.\n\nA particular characteristic of the archaeological study of SFP is that they leave little room for easy or even fantastic interpretations of historical events. When we are not very clear about the story or when it has several gaps, SFP force us to base our interpretations on data and make our assumptions explicit. This is where, to our view, ABM can be a fundamental tool for archaeologists interested in SFP. Not only does ABM allow us to make the experiment repeatable, it requires us to make our assumptions explicit by defining agents, variables, processes, indicators, scenarios, and hypotheses in a conceptual model. In this paper, we presented such a conceptual model about the SFP of Somers, which is not intended to be definitive in any way, but rather the first implementation of a tool that allows testing hypotheses in order to tell a story more or less reliable, with firm supports, about the Somers shipwreck.\n\nAs stated earlier, we based our ABM on the theoretical models of maritime archaeologists Keith Muckelroy and Martin Gibbs. In his seminal article on the cultural aspects of shipwreck SFP, Gibbs referred to further developments on various aspects of Muckelroy’s model stating that:\n\n“While both Souza’s (1988) and O’Shea’s (2002) studies make significant advances in our understanding of cultural ‘scramblers’ and ‘extracting filters’, it could be argued that the ‘pre-depositional’, ‘depositional’, ‘post-depositional’ structure is still primarily oriented towards explanation of the archaeological deposition and distribution, rather than the cultural processes behind them. [...] I would suggest that the alternative way of approaching shipwrecks as cultural processes is to structure our understanding around the nature of the event and the sequence and range of potential responses at each stage.”13\n\nIn our opinion, the fact that Gibbs considers Muckelroy’s approach and the subsequent developments by Souza and O'Shea as limited in their ability to explain cultural processes is because he may be asking for the impossible. That is, he is hoping that Muckelroy’s model will provide a historical/social explanation of a shipwreck SFP when it is not necessarily trying to do so. The same argument could be used in the opposite direction. It could be said that Gibbs’ model does not explain the deposition, degradation, and distribution of elements of a shipwreck; but it does not do so because that is not its objective. Rather, it seems that the Muckelroy and Gibbs models bring two different levels of explanation to understanding the shipwreck SFP: one the time’s cycle, the other the time’s arrow.\n\nThe time’s cycle and arrow metaphors come from Stephen Jay Gould’s homonym book (Time’s Arrow, Time’s Cycle, 1987). This text is cited by Edward Harris in his Principles of Archaeological Stratigraphy (1989) to illustrate two aspects of the study of stratification in an archaeological site: the cyclical and the historical aspects.68 The archaeological stratification of a site, with its stratification units (deposits and interfaces), represents the time’s cycle insofar as its formation is always the result of “the same, repetitive processes, i.e. deposition or degradation”.68 Consequently, they are timeless and universal, being found in any archaeological site in the world. The interpretation of the structure of the site and its artefactual content provide the time’s arrow, the historical aspect of the stratification. “Without an appreciation of the difference between the two bodies of data that represent time's arrow and time's cycle, the unique event from the repetitive process, it will be difficult for an archaeologist to understand, record and interpret archaeological stratification”.68\n\nFrom this distinction between time’s cycle and time’s arrow, the apparent discrepancy between Muckelroy and Gibbs’ models could simply be that they each address different aspects of the SFP. In our opinion, Muckelroy’s model and its subsequent extensions are more oriented towards the cyclical, timeless aspects of the process including environmental factors, extracting filters, and scrambling devices, as well as the effects they produce, which we will find in all shipwrecks, places and times. Gibbs’ model addresses primarily the historical, temporal aspects of the SFP such as the motivations and decisions of different people in different places and at different times. In this sense, both models do not seem opposite but totally complementary. One provides the how of the story, the other the why. Hence, the ABM we developed integrates components of both theoretical models.\n\nA fundamental principle when creating an ABM is to start simple, with the fewest possible number of elements (agents, variables, processes) that would allow us to answer our research questions. Since we based our ABM on the theoretical models of Muckelroy and Gibbs, whose general characteristics were conceived to be applicable to any shipwreck, it contains the fewest possible agents, processes, and indicators to explore their SFP, and should be suitable for other shipwrecks without major modifications. However, in its current version, our model is specific to the Somers and was designed to explore specific questions about this particular shipwreck based on its story. Therefore, for our ABM to be used in other case studies, some extensions to the conceptual model would be necessary.\n\nRegarding the model’s processes that simulate environmental aspects of a shipwreck SFP, they should be extended to consider the hydrodynamic effects of high or low energy conditions, which differentially modify the shipwreck through physical or chemical-biological erosion processes.18 Some of these effects are considered, for example, in the fluid dynamics simulations carried out by maritime geophysicist Rory Quinn.\n\nHowever, the central role assigned to fluid dynamics in Quinn’s studies of shipwreck SFP is based on “the acceptance that physical processes dominate early-stage wreck site formation”.25 This “acceptance” may be rather obvious from his perspective as a geophysicist, given that the interactions of the shipwreck-current-sediment start when the ship is deposited on the seabed, which may seem to be where the process begins. But it is not so clear from an archaeological point of view, which considers that the SFP begins long before the deposition of the ship and that it not only involves physical processes but also a series of decisions by various agents at different moments of the process, such as those considered by Souza and Gibbs,11,13 which modify the configuration of the shipwreck in its entirety. From this perspective, it is apparent that physical processes constitute one of many components of the SFP, not necessarily the dominant one.\n\nRegarding the wrecking process, two necessary extensions for the ABM would be needed to simulate 1) catastrophic wrecking, and 2) the sinking process. The first extension would allow simulating different wrecking scenarios, such as grounding and battle, where the ship would have undergone serious structural damage. We did not define such processes in our conceptual model since none of these happened to the Somers, which sank after capsising due to weather conditions. Because of this, even though the setup controls allow for the selection of initial conditions that would modify the ship’s configuration (jettisoning, cutting masts, holes on the hull), simulations for the Somers’ SFP would start with the ship rather intact, settled on the seabed.\n\nHowever, even in the absence of major structural damage, the sinking process itself can potentially alter elements’ disposition within the ship and, consequently, the seabed distribution of the shipwreck, due to spatial rearrangements during the transit from surface to bottom. This would be especially true for ships sinking in deep waters, such as the Somers. Therefore, the ABM should be extended to simulate the sinking process. This would allow for the exploration of the effect of variables such as cargo and ballast distribution, nautical manoeuvres, list, and position of the ship when deposited on the seabed. Such an extension could also be designed to allow for simulating wrecking scenarios with different depths to explore, not only the aforementioned effects of the sinking process, but also if shallower depths would increase the probability of pieces disappearing during salvage operations.\n\nThe Salvage process could also be refined in future versions of the ABM. In its current configuration, all salvage operations are equivalent in terms of the number and type of objects that can be removed from the environment as a result of each operation. This is because the probability of object disappearance is a function of the value assigned to them, which we assigned arbitrarily and remains constant throughout the simulation time. This value assignment is probably inaccurate in historical terms, but we assumed it that way in order to simplify the ABM. In future versions of the model, value assignment could be refined so that it is not fixed from the start, but rather reflects the different interests of different actors at different times, along the SFP. For example, a sailor would probably not have been interested in salvaging an artillery piece from the wreck in the event of a survivor salvage operation. Therefore, the piece’s value during said operation would be very low. But the opposite could be true if it were the Commodore who planned a systematic salvage operation, then artillery could be most valuable to the rescue.\n\nAdditionally, the Salvage subprocess is executed only on those elements categorised as mobiles and accessories. The four element categories (major structural, minor structural, fixtures, and cargo) were taken directly from Gibbs, who proposed them as a way to “simplify and characterise the components [...] based on the relative ease with which items can be removed and how they relate to the structural integrity of the vessel”.13 However, he also pointed out that this categorisation of elements is “flexible and not strictly hierarchical”.13 For example, a component of the cargo located in the bottom of the ship would be assigned category 1 (cargo) but could be much heavier or more difficult to access than a fixture (category 2) or structural element (categories 3 and 4) located elsewhere in the ship. From our point of view, the possible conceptual limitation of element categorisation that Gibbs observes is completely avoided in our ABM by being based on a 3D environment and programmed so that processes involving element removal are not based merely on item category but rather on a salvage difficulty calculation, which involves both the item’s mass (in turn affected by degradation) and its three-dimensional location.\n\nIn addition to salvage operations, the model should also be extended to include other intrusive operations. We refer in particular to archaeological operations, particularly excavation, which constitute an important part of a shipwreck SFP as they profoundly modify the shipwreck’s element content and spatial disposition.18 As with other conditions, we did not include such operations in the current version of our ABM, since no intrusive archaeological operations have been carried out so far in the Somers shipwreck.\n\nFinally, using ABM would potentially allow archaeologists to explore how much of a shipwreck’s SFP can we know if we were only to perform a surface, non-intrusive recording, that is, without excavating the shipwreck. With this in mind, we added to the ABM an additional component that is obviously not found in any of the theoretical models: the degradation and deposition sequences. Traditionally, these could only be obtained from the archaeological records of a stratigraphic excavation; however, with ABM this is no longer a limitation. By integrating the analysis of such sequences into the analysis of the SFP we could refine our understanding of its cyclical parts, thus refining our timeline and the story we tell.\n\n\n5. Conclusions\n\nIn this paper we have tried to provide a glimpse of the scope ABM could achieve as a tool for the archaeological study of site formation processes, specifically shipwrecks. Although this type of modelling, has been increasingly used in archaeology as a methodology for analysing complex systems, to our knowledge, this is the first time it has been applied for studying SFP.\n\nThrough ABM it was possible to integrate two different but complementary theoretical models, and several sources of information as diverse as 19th-century shipbuilding plans and specifications, eyewitness accounts, and official naval reports from the time; but also contemporary oceanographic, geophysical, and archaeological data. All of these were coherently and interrelatedly integrated into a model designed for analysis and experimentation.\n\nThe fundamental contribution that our modelling proposal gives to the archaeological discipline, in general, is the possibility of proposing a wide variety of hypotheses and carrying out controlled and repeatable experiments that were not possible before. This directly impacts our ability to analyse and interpret the archaeological context and, thus, the ultimate goal of the discipline, which is to answer questions about the past. In the field of maritime archaeology, in particular, our proposal contributes to providing the discipline with a methodological tool that, through the integration of conceptual models, historical sources, and archaeological data, allows for generating sustained interpretations of shipwrecks and their site formation process.\n\n\nAuthor contributions\n\nRVS: Conceptualisation, Data Curation, Formal Analysis, Investigation, Methodology, Software, Supervision, Validation, Visualisation, Writing – Original Draft Preparation, Writing – Review & Editing.\n\nJMH: Data Curation, Formal analysis, Funding Acquisition, Investigation, Methodology, Project Administration, Resources, Writing – Review & Editing.",
"appendix": "Data availability\n\nVega-Sánchez R. Supplementary material for “Agent-based modelling for the study of shipwreck site formation processes: A theoretical framework and conceptual model”. OSF; 2022, DOI 10.17605/OSF.IO/2H97R. Available from: https://osf.io/2h97r/ 70\n\nThis project contains the following extended data:\n\n• Supplementary material 1. Degradation and corrosion equations.\n\n• Supplementary material 2. Coral adherence/growth.\n\n• Supplementary material 3. An alternative for coding the movement process of mobile agents.\n\nExtended data are available under the terms of the terms of the Creative Commons Attribution 4.0 International (CCBY 4.0).\n\n\nAcknowledgements\n\nFor their most valuable comments and suggestions to both the project and manuscript, we sincerely thank:\n\nDr Rodrigo Ortiz Vazquez (Centre for Maritime Archeology, University of Southampton, UK)\n\nDr Ignacio Rozada (1QB Information Technologies, Vancouver, Canada)\n\nDr Nicolás Ciarlo (CONICET-Instituto de Arqueología, Universidad de Buenos Aires Argentina)\n\nProf Patricia Murrieta-Flores (Digital Humanities Centre, Lancaster University, UK)\n\nWe are also grateful to Margherita M. Desy (Historian, Naval History and Heritage Command Detachment Boston/USS Constitution, USA), Sandra L. Fox (Supervisory Librarian, Reference Section, Library, Naval History and Heritage Command, USA), and Kate Monea (Manager of Curatorial Affairs, USS Constitution Museum, USA) for their selfless and expeditious help in finding information on the use of metal sheathing on 19th-century US Navy ships.\n\nFinally, we thank Guadalupe Rozada and Regina Vega for their constant support and valuable feedback throughout the project.\n\n\nReferences\n\nMuckelroy K: Maritime archaeology London:Cambridge University Press;1978.\n\nGibbs M, Duncan B:Cultural Site Formation Processes Affecting Shipwrecks and Shipping Mishap Sites.Keith ME, editor. Site Formation Processes of Submerged Shipwrecks Gainesville:University Press of Florida; 2016; pp. 179–207. Publisher Full Text\n\nClarke DL: Analytical Archaeology. London:Methuen & Co.;1968.\n\nMitchell M: Complexity: a guided tour. New York:Oxford University Press;2009.\n\nWilensky U, Rand W: An Introduction to Agent-Based Modeling: modeling natural, social and engineered complex systems with NetLogo. Cambridge:MIT Press;2015.\n\nRand W: Introduction to Agent-Based Modeling (Summer 2017): When should you use ABM? Complexity Explorer.2017. Accessed 2017 Nov 20.Reference Source\n\nHerrera JM, Jiménez P, Pacheco Ruiz R, et al.:La memoria anfibia: arqueología marítima de la guerra entre México y los Estados Unidos, 1846-1848.Landa CG, Hernández de Lara O, editors. Arqueología en campos de batalla: América Latina en perspectiva Buenos Aires:Aspha Ediciones; 2020; pp. 63–116.\n\nSchiffer MB: Archaeological Context and Systemic Context. Am. Antiq. 1972; 37(2): 156–165. Publisher Full Text\n\nDumas F: Deep-Water Archaeology. 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Antropología, complejidad, sensoramiento remoto y SIG en la arqueología marítima.Tesis de Licenciatura en Arqueología, Escuela Nacional de Antropología e Historia.2001.\n\nHerrera JM, Buffa V, Cordero A, et al.: Maritime Archaeology in Uruguay: Towards a Manifesto. J. Marit. Archaeol. 2010 Oct 1; 5(1): 57–69. Publisher Full Text\n\nHerrera Tovar JM: Proyecto Arqueológico: Arqueología marítima de la Guerra de Intervención. Ciudad de México:Instituto de Investigaciones Antropológicas, UNAM;2016; 32.\n\nPreiser-Kapeller J:Harbours and Maritime Networks as Complex Adaptive Systems – a Thematic Introduction.Preiser-Kapeller J, Daim F, editors. Harbours and Maritime Networks as Complex Adaptive Systems. RGZM; 2015; pp. 1–23. Reference Source\n\nOrtiz VR: An integrated methodology to study site formation processes on submerged shipwrecks in the 21st c [PhD]. University of Southampton;2018. Accessed 2021 Mar 7.Reference Source\n\nHerrera Tovar JM: The reflexive navigator. Theory and directions in maritime archaeology [Doctor of Philosophy]. Adams J, editor.University of Southampton; 2008.\n\nBinford LR: Archaeology as anthropology. Am. Antiq. 1962; 28(2): 217–225. Publisher Full Text\n\nDean JS, Gumerman GJ: Understanding Anasazi culture change through agent-based modeling. … -based modeling of ….1998; p. 32.Reference Source\n\nAxtell RL, Epstein JM, Dean JS, et al.: Population growth and collapse in a multiagent model of the Kayenta Anasazi in Long House Valley. Proc. Natl. Acad. Sci. 2002; 99(S3): 7275–7279. PubMed Abstract | Publisher Full Text Reference Source\n\nWurzer G, Kowarik K, Reschreiter H: Agent-based Modeling and Simulation in Archaeology. Cham:Springer;2015. Publisher Full Text\n\nQuinn R: The role of scour in shipwreck site formation processes and the preservation of wreck-associated scour signatures in the sedimentary record – evidence from seabed and sub-surface data. J. Archaeol. Sci. 2006 Oct 1; 33(10): 1419–1432. 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Seventh ed.Edinburgh:Adam and Charles Black;1841; pp. 90. Publisher Full Text\n\nSloat JD: Stowage of Hold U.S. Sloop Vandalia. 1849. (U.S. National Archives and Records Administration, 107-14-2C).\n\nStowage of the Water Casks and Kentledge of the U.S. Sloop of War Marion. ca. 1840. (U.S. National Archives and Records Administration, 107-14-6A).\n\nNeyland RS, Brown HG: H.L. Hunley recovery operations Washington, D.C.:Naval History and Heritage Command;2016.\n\nSilverstone PH: The Sailing Navy, 1775-1854. Annapolis:Naval Institute Press; 2001; 101 p. (The U.S. Navy Warships). Reference Source\n\nWegner DM: Fouled Anchors: The Constellation Question Answered. David Taylor Research Center; 1991. Reference Source\n\nO’Scanlan T: Diccionario Marítimo Español. Madrid:Imprenta Real;1831. Reference Source\n\nDana RH Jr: The Somers, by a Seaman. Pennsylvania Inquirer and National Gazette;1843 Jan 17; 1.\n\nLogbook of the Mississippi 1846.(The U.S. National Archives and Records Administration (NARA I), RG24 118 18w4/6/06/02).\n\nAlam J, Das AK, Rahman MM, et al.: Effect of waterlogged condition on wood properties of Acacia nilotica (L) Debile tree. Bangladesh J. Sci. Ind. Res. 2015 Jul 30 [cited 2019 Oct 26]; 50(2): 71–76. Publisher Full Text Reference Source\n\nDel Castillo Sarabia LE: Macrofauna bentónica en fondos blandos del Arrecife Hornos, Parque Nacional Sistema Arrecifal Veracruzano, México [Maestría en Ciencias (Biología Marina)]. Granados Barba A, editor.UNAM; 2007.\n\nSánchez PS: Descripción de perfiles estratigráficos en campo y Análisis físico-químico de suelos y sedimentos INAH / ENAH;2005.\n\nFolk RL: Petrology of Sedimentary Rocks. Hemphill Publishing Company;1980; 182. Reference Source\n\nBowens A: Underwater archaeology: the NAS guide to principles and practice. Society NA, editor.Oxford:Blackwell Pub;2009.\n\nLópez Garrido PH, González-Sánchez J, Escobar BE: Fouling communities and degradation of archeological metals in the coastal sea of the Southwestern Gulf of Mexico. Biofouling 2015; 31(5): 405–416. PubMed Abstract | Publisher Full Text\n\nRailkin AI: Marine Biofouling: Colonization Processes and Defenses. CRC Press;2004; 303. Reference Source\n\nThornhill DJ, Rotjan RD, Todd BD, et al.: A connection between colony biomass and death in Caribbean reef-building corals. PLoS One 2011 Dec 22; 6(12): e29535. PubMed Abstract | Publisher Full Text\n\nDomínguez Castanedo N d C: Estudio de la macrofauna bentónica de la laguna arrecifal de Isla Sacrificios, Veracruz [Maestría en Ciencias (Biología Marina)]. Solís Weiss V, editor.UNAM;2007.\n\nGonzález Flores E: Variación intraespecífica en la composición isotópica y elemental de especímenes de Xestospongia subtriangularis perteneciente al Sistema Arrecifal Veracruzano (SAV) [Maestría en Ciencias (Biología Marina)]. Escobar Briones E, editor.UNAM; 2009.\n\nRivera Ramírez F: Determinación de metales traza (Cu, Ni, Pb, V y Fe) en agua de mar y en dos especies de corales en el Arrecife Sacrificios del Sistema Arrecifal Veracruzano (SAV) [Maestro en Ciencias (Química Acuática)]. MTL RH, editor.UNAM; 2006.\n\nSalas Pérez J d J, Salas-Monreal D, Monreal-Gómez MA, et al.: Seasonal absolute acoustic intensity, atmospheric forcing and currents in a tropical coral reef system. Estuar. Coast. Shelf Sci. 2012 Mar 20; 100: 102–112Publisher Full Text Reference Source\n\nPournou A, Jones AM, Moss ST: Biodeterioration dynamics of marine wreck-sites determine the need for their in situ protection. Int. J. Naut. Archaeol. 2001 Dec 1; 30(2): 299–305. Publisher Full Text Reference Source\n\nBjördal CG, Nilsson T: Reburial of shipwrecks in marine sediments: a long-term study on wood degradation. J. Archaeol. Sci. 2008 Apr 1; 35(4): 862–72. Publisher Full Text http://www.sciencedirect.com/science/article/pii/S0305440307001239\n\nHerrera Tovar JM, Jiménez P, Rodríguez ÉS, et al.: Proyecto Arqueológico Arqueología Marítima de la Guerra de Intervención (1846-1848). Informe Año 2 (2018-2019). Instituto de Investigaciones Antropológicas, UNAM;2019 Jul.\n\nBjördal CG: Microbial degradation of waterlogged archaeological wood. J. Cult. Herit. 2012 Sep 1; 13(3, Supplement): S118–S122. Publisher Full Text Reference Source\n\nMcCawley JC: Waterlogged artifacts: The challenge to conservation. Journal of the Canadian Conservation Institute. 1977; 2: 17–26. Reference Source\n\nBorges LMS: Biodegradation of wood exposed in the marine environment: Evaluation of the hazard posed by marine wood-borers in fifteen European sites. Int. Biodeterior. Biodegradation 2014 Dec 1; 96: 97–104. Reference Source\n\nRobinson WS: The corrosion and preservation of ancient metals from marine sites. Int. J. Naut. Archaeol. 1982 Aug; 11(3): 221–231. Publisher Full Text\n\nSanders D: Knowing the Ropes: The Need to Record Ropes and Rigging on Wreck-Sites and Some Techniques for Doing So. Int. J. Naut. Archaeol. 2010 Mar; 39(1): 2–26. Publisher Full Text\n\nJenssen V:Conservation of wet organic artefacts excluding wood.Pearson C, editor. Conservation of Marine Archaeological Objects. Oxford:Butterworth-Heinemann; 1987; pp. 122–163. Reference Source\n\nPeacock EE: Characterization and simulation of water-degraded archaeological textiles: a review. Int. Biodeterior. Biodegradation 1996 Jan 1; 38(1): 35–47. Publisher Full Text Reference Source\n\nWoods Hole Oceanographic Institution, United States:Navy Dept. Bureau of Ships. The Fouling of Metallic Surfaces. Marine fouling and its prevention; prepared for Bureau of Ships, Navy Dept Woods Hole, MA:United States Naval Institute; 1952. pp. 349–364. Reference Source\n\nHarris EC: Principles of Archaeological Stratigraphy. 2a ed.London:Academic Press;1989.\n\nBarker P: Techniques of Archaeological Excavation. London and New York:Routledge;1996; 285p. Reference Source\n\nVega-Sánchez R: Supplementaty material for ‘Agent-based modelling for the study of shipwreck site formation processes: A theoretical framework and conceptual model’. Open Science Framework. 2022. Accessed 2022 Sep 27.Reference Source\n\nLuce SB: Seamanship: Compiled from Various Authorities and Illustrated with Numerous and Selected Designs, for the Use of the United States Naval Academy. New York:D. Van Nostrand;1868.\n\nBiddlecombe G: The Art of Rigging. London:Charles Wilson;1848; 136p. Reference Source\n\n\nFootnotes\n\n1 As aforementioned, pseudocode does not refer to a formal coding language. It is rather “a midway point between natural language and formal programming language [that] can be read by anyone, regardless of his or her programming knowledge, while, at the same time containing algorithmic structure that makes it easier to implement directly into real code”. 5"
}
|
[
{
"id": "165087",
"date": "23 Mar 2023",
"name": "Katherine A. Crawford",
"expertise": [
"Reviewer Expertise Archaeology",
"simulation",
"computational archaeology"
],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address 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 proposes a new approach for studying the shipwreck site formation process (SFP) in maritime archaeology, using a complex adaptive system (CAS) approach and agent-based modelling (ABM). The authors argue that while previous SFP models based on systemic theory have been valuable, they fall short in explaining the distribution of elements in a shipwreck. By applying the principles of CAS and ABM, the authors suggest that it is possible to better understand the interactions between various social and natural elements in the formation of an archaeological site, using the 19th-century USS Somers as a case study. A detailed overview is then provided about the different components of an ABM that could be applied to address the authors' research questions.\nOverall, the article and methods are well thought out and provide a novel approach to addressing the challenges of studying SFP. The different processes and components of the model are well-described and easily understood. This will be an incredibly useful paper on how to conceptualize a model for future implementation, especially on a topic that has yet to receive any ABM attention. However, to improve the readability and overall flow of the paper, I recommend the following structural changes and additions, detailed by subheading.\nSection 3 Results: this section would be more appropriately termed Theoretical Model Description since the authors are not presenting results of the actual outcomes of the model. I would also recommend that a brief paragraph description be given of the model and what it will do prior to outlining the specific components beginning in section 3.1. I would also recommend revising the overall model subsections to follow the ODD protocol (Grimm et al. 2010. The ODD protocol: a review and first update. Ecological Modelling 221: 2760-27681). Adaptation of an ODD will also make it easier to follow the logic of the different model components discussed. For instance, Section 3.1.1 falls under the agents subheading, but it is unclear if this is being used as an overall model environment or if the environment is being used as an agent and how. I recommend that the authors re-think if all of the components are actually agents or model environment parameters and potentially add a specific sub-section on environment parameters which is currently missing from the model description. Adjustment to an ODD model description will make it easier for the reader more fully comprehend the proposed model design and would follow the standard approach for describing archaeological models.\nSection 3.3 Processes: it is unclear why pseudocode is provided for some subprocesses and not others. Given the length of the paper, I question the necessity of including the pseudocode sections. I urge the authors to consider including the full pseudocode within supplementary material and only mention the relevant processes. This would also allow for a better understanding of how the model will be run without having to refer to individual sections within the article.\nSection 3.4 indicators and their analysis: a general overview of the different validation datasets and techniques should be included prior to going into detail in the subsections. 3.4.2 goes into detail about how the simulated dataset will be validated against real-world data, however, the other subsections do not. Alternatively, a discussion of the model’s validation could be better suited to be expanded upon in the discussion section, see below.\nDiscussion section: the first part of the discussion section would be better suited in the introduction (1.1.4) where the authors discuss ABM as a tool for addressing their research questions. Notable is the absence of a discussion on how the model will be critically validated and what methods of analysis will be used. I recommend that the authors also think about how changes to the model will need to be done if validation is inaccurate. The authors have provided a comprehensive discussion of the benefits of applying ABM to their research questions; however, this section would be improved by addressing the limitations of the approach, and discussing how these might be mitigated by other approaches or analyses. Additionally, the authors have also not stated if this model will actually be created or if the aim is to just present the theoretical model concept for potential implementation by other archaeologists.\nSome other minor points:\np.7 (of the pdf version of the article) further description of why ABM is more appropriate than other methods such as fluid dynamic models is needed. It’s still not completely clear why ABM should be applied in this case study.\n\nSome of the figures need to be included at a higher resolution. Figure 8 needs to be amended so that the text is all readable; Figure 11 text is a bit blurry; Figure 13 is quite blurry, if possible, I recommend a higher resolution image.\n\nIn the general description of the ABM on p. 14, additional commentary on how the static and dynamic agents were chosen as the most relevant for inclusion in the present model is needed.\n\nIs the work 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? Partly",
"responses": []
},
{
"id": "175763",
"date": "13 Jun 2023",
"name": "Nicolás C. Ciarlo",
"expertise": [
"Reviewer Expertise Maritime and nautical archaeology",
"historical metallurgy",
"and archaeometry"
],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 statement about the manuscript:\nThe article is well-written and organised and provides an insightful archaeometric approach to shipwrecks interpretation, with an emphasis on the application of a novel and useful tool for better understanding the site formation process (SFP).\nI think this proposal is very exciting. To conceive shipwreck SFP as complex adaptive systems (CAS) provide scholars with a powerful heuristic tool (in this case, agent-based modelling – ABM) to obtain non-linear and multi-causal answers about complex behaviours comprising both human and natural factors and dynamics. Furthermore, considering the observable elements of a site as an emergent phenomenon that result from the interactions and changes developed over time between different agents, and addressing how the behaviour of agents led to a particular result (a pattern in the archaeological record) are both original approaches that could be applied to the analysis of SFP of other shipwrecks. With a high degree of novelty in the field of nautical archaeology, this study applies a methodological approach of computational modelling and simulation (ABM tool) to attain shipwreck SFP, which is very valuable considering the inner limitations that the documentary and archaeological information usually present about a particular ship’s life and environmental and cultural conditions that surrounded it through time.\nThe methodological procedure to develop the ABM code is well-developed. Also, the information used to feed this model is clearly presented and detailed. For instance, the nautical features of the USS Somers, needed to define the 3D model of the ship, were defined based on previous solid research on documentary evidence, an archaeological non-intrusive record of the site, and proxy data from other contemporary US naval vessels. The different elements that comprise the conceptual model used by the authors, their main characteristics and structure, are also explained. Nonetheless, I suggest minor amendments in some individual attributes or properties of a few agents to improve their reliability (see below).\nThe sections “3. Process” and “4. Indicators” are well-explained and justified (I went through the statistical features of pseudocode for different operations, from 3.3.2.1 to 3.3.2.5), yet minor changes are also suggested (see below). The section “5. Degradation”, I think it could be merged into a previous section. The section “6. User interface” is clear, yet an image on the interface controls would be helpful to better understand it.\nAs it is stated in the discussion, this research developed and applied a powerful heuristic tool to test different situations under different and variable conditions to assess the SFP of the USS Somers and provide a series of possible histories to be then tested against the archaeological record, thus contributing to both the time’s arrow and time’s cycle and gaining a deeper and more reliable understanding on the actual natural and cultural processes to which it was subjected through time.\nThe manuscript is accepted for indexing after minor revisions on the aspects detailed below are developed.\nPlease, consider replacing the following expressions or terms:\nSection 2.2.1: metal lining covering the bottom for metal (or copper) sheathing.\n\nSection 2.2.1: rudder blade for rudder (also, see its mention in other sections of the manuscript).\n\nSection 2.2.1: water lines for waterline.\n\nSection 2.2.1: ¿what do covers stand for? ¿do you mean decks or deck planks?\n\nSection 2.2.1: tubular metal stock for metal stock or movable iron stock.\n\nSection 2.2.2: the hold located on the bottom of the hull for the hold (the clarification is unnecessary)\n\nSection 3.1.2: stern foot for stem foot.\n\nSection 3.1.2: bullets for shot.\n\nPlease, revise the following expression:\nSection 2.2.1: stanchions that supported both covers, knees, beams that supported the lower deck…\nPlease, revise the following statements:\nSection 2.2.1: “Anchors were probably of the “Old Pattern Admiralty Long Shank” type, adopted by the British navy at the end of the 18th century and in use for much of the 19th century”. The information on the use of this type of anchor is incorrect. By the time UN Navy built and equipped the USS Somers, it is most likely that one of the new models patented since the 1820s was used for that purpose. Please, revise a more specialized and precise bibliography on this theme (see Curryer, 1999; Ciarlo, 2019, 2023, and references therein). To consider this and other technical comments would be significant for authors to better define the individual properties of agents that make up the ABM.\n\nSection 2.2.1: “wrought iron density = 484 pounds/ft3)”. The density of cast iron should be considered for pig iron ballast, which was made of this material. It ranges from about 6.9 to 7.4 g/cm3 (among cast iron, white iron in particular has the higher value of density).\n\nSection 2.2.1: “distributed along the centre of the hold”. Pig iron (small ingots) was also distributed aft and fore the ship’s central area (see Carabias et al., 2023).\n\nSection 3.1.1: “ourABMstarts with the ship already settled on the seabed, that is, the model does not take into account the transit from the water surface to the bottom. Although this transit could certainly have altered the distribution of elements of the ship, in this first version of the model we assume that such alteration was minimal”. This can be applied with a degree of confidence in this case; although, other wreck events (e.g. in a blast or fire) comprise a high disturbance of the structure, equipment, and other elements between the two stages. Depth and drift of the remains during sinking should also be considered here.\n\nSection 3.1.2: “…for the ABM we considered that wood is already saturated at the beginning of the simulation”. It probably took a while for a ship’s timber to be fully saturated with water. Considering the dynamic potential of the ABM proposed by authors, I think this change over time should also be considered in order to apply the model.\n\nSection 3.1.2: “The nails used to fasten the linings were usually made of copper, zinc, or tin alloy”. As a general statement, one can pose that at that time wooden vessels sheathed with copper (or brass sheets) were fastened with copper or copper-based fastenings (e.g. bronze bolts and bronze nails) below the waterline. Above it, most fastening and other metal fittings were made of iron, given the former was more expensive. This scheme seems appropriate to the case of USS Somers (see McCarthy, 2005; Ciarlo, 2017, 2023; and references therein).\n\nSection 3.1.2: “Creuze’s mention that the nails were not made of pure copper but of an alloy of zinc, copper, and tin may suggest the use of naval brass. Therefore, for ABM we assumed that the latter was used for the hull sheathing”. This assumption is not correct. Bolts and other fastenings were indeed made of naval brass, but sheathing planks were made either of unalloyed (pure) copper or brass (the latter, with no addition of tin, as they were manufactured by rolling) (see the suggested bibliography).\n\nSection 3.1.2: “…chains, anchors, and carronades are cast iron”. Despite guns, both chains and anchors were manufactured with wrought iron.\n\nSection 3.1.2: “Hull sheathing is naval brass”. This should also be changed for the analysis. We actually know that the sheathing of the USS Somers was made of pure copper (as well as the sheathing of other US naval vessels of the time such as the USS Constitution).\n\nSection 3.1.2: “The salvage-value attribute is a continuous numerical variable whose initial values were assigned to each agent arbitrarily. We assumed that, similar to the previous example, artillery, ammunition, or some equipment would be more valuable for salvaging than structural elements”. I think it rather depends on the situation of the wreck and the survivors’ necessities. If they were compelled to build a camp or another vessel to sail away and survive, timbers would have been most valuable for them.\n\nSection 3.1.2: “The degradation-percentage attribute is a continuous variable”. It would be interesting to add a correction value depending on the environmental conditions where the wreck site is located, or at least to mention the possible variations that can be expected if different water conditions are faced.\n\nSection 3.3.2.3: “…models of shipwreck SFP should include at least some rigging elements. However, we decided not to include these elements nor their degradation process in the Somers”. The two reasons why authors decided not to include rigging (lack of precision in documentary sources and lack of experimental data) seem not sufficient given these drawbacks did not hinder them to include in the model other ship’s elements for which they had not this information either. The authors mentioned that experimental data for wood and metal is available, but this information is of a general nature. Differences between ferrous and non-ferrous alloys are considered (iron versus brass), but no distinction is established within ferrous alloys, e.g. differences between wrought and cast (and within the cast iron, between grey and white). For the sake of analysis, is it not possible to include rope within the wood category (or a general one, for organic materials)?\n\nSection 3.4.4. “In terms of the historical explanation of the SFP, another necessary indicator is the number of salvage operations carried out on the shipwreck, both contemporary (systematic) and after the sinking (opportunistic)”. As no documentary data are available about the first three types of salvage operations defined by Gibbs, how is it possible to establish a reliable criterion for these activities after the sinking?\n\nSection 3.7. “the accounts of the wreck of the Somers do not mention that the anchors were dropped but they do mention that the boat was launched; there is also no mention of crisis or systematic salvage operations. These data should remain constant in all simulation scenarios”. That a certain action is not mentioned in the documentary sources does not mean that it actually did not take place in the past. At least in the case of actions that likely occurred during a sinking event such as the one experienced by the USS Somers, I think they should also be tested (as well as activities for which no records are available).\nI suggest considering the following works:\nOn the background on SFP of shipwrecks:\nFernández-Montblanc et al. (2016) Fernández-Montblanc et al. (2018a) Fernández-Montblanc et al., (2018b) Ortega Pérez et al. (2023)\nOn anchors (particularly, the Old Admiralty Long Shank Pattern and other types used during the 19th century):\nCiarlo (2019) Ciarlo (2023) Curryer (1999)\nOn pig iron, a recent historical and archaeological detailed account of its use in mid-19th century ships is provided by:\nCarabias et al. (2023)\nOn metal fittings and fastenings of late 18th to mid-19th century wooden vessels with copper sheathing, see:\nCiarlo (2017) Ciarlo (2023) McCarthy (2005)\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": []
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https://f1000research.com/articles/11-1525
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https://f1000research.com/articles/11-840/v1
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27 Jul 22
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{
"type": "Research Article",
"title": "In silico and in vitro analysis of THCA synthase gene in Moroccan Cannabis Sativa, L",
"authors": [
"Fadwa Badrana",
"El Mostafa El Fahime",
"Abdelrhani Mokhtari",
"Abdelmajid Soulaymani",
"Najete Safini",
"Bouchra Chaouni",
"Gabriel Malka",
"Hamid El Amri",
"El Mostafa El Fahime",
"Abdelrhani Mokhtari",
"Abdelmajid Soulaymani",
"Najete Safini",
"Bouchra Chaouni",
"Gabriel Malka",
"Hamid El Amri"
],
"abstract": "Background: Cannabis (Cannabis sativa, L.) is an archaic, most infamous plant with unique therapeutic characteristics, and great economic interest. The identification of its THCAS synthesizing enzyme was a great leap forward in Cannabis investigation. However, basic molecular biology for THCAS gene expression remains largely unstudied. Methods: In this paper, we explored the ability of bioinformatics resources in the design of molecular cloning protocol and prediction of three-dimensional structure of recombinant THCAS in Moroccan Cannabis sativa variety. We further described conventional experiments performed in vitro. We used specific markers to isolate the nucleotide sequence of THCAS from the leaves of Cannabis, without including the native signal sequence. Results: As a result, we found high sequence similarity with THCAS and the mRNA precursor of the same gene as previously reported. In addition, we predicted the structure of the recombinant THCAS using the organic crystal structure prediction method, the amino acid sequence reference, and the artificial intelligence technology. Conclusions: This study paves the way to successfully express recombinant THCAS enzyme in hosts like Pichia pastori.",
"keywords": [
"Cannabis Sativa",
"THCAS",
"molecular cloning",
"bioinformatics",
"modeling."
],
"content": "Introduction\n\nCannabis sativa L., basically originated from Central Asia, is the most widely used illegal drug around the world, appallingly consumed by around 160 millions individual. It is certainly the most popularly cultivated with two main derivatives, which are marijuana (الكيف) and resinالحشيش) 1). In fact, Morocco remains the world’s leading producer of cannabis resin as declared by the United Nations Office on Drugs and Crime (UNODC).2 Remarkably, since the psychoactive cannabinoid tetrahydrocannabinol (THC) was first discovered in 1964 and the human endocannabinoid system in 1980, many studies focused on the therapeutic effects and pharmaceutical applications of THC. Currently, the active ingredient THC is increasingly used in the treatment of nausea and vomiting associated with chemotherapy, loss of appetite due to AIDS, pain, and muscle spasms because of multiple sclerosis. As of today, Sativex, a cannabis-based preparation containing THC, was licensed in Canada as a neuropathic analgesic for multiple sclerosis.3 Furthermore, many other applications are still being explored, and demand for Pharma-Grade THC continues to grow.4 The heterologous production of cannabinoids or their precursors has been attempted by different microorganisms, such as E. coli,5–9 Zymomonas mobilis,10 Synechocystis spp.11 and Synechococcus elongatus.12 Besides, fungal species like Saccharomyces cerevisiae,13–17 Kluyveromyces marxianus,13 Komagataella phaffii,14,18,19 Candida viswanathii,20 or plant species like Chlamydomonas reinhardtii12 and Nicotiana benthamiana were also exploited.21–23\n\nNew disquisitive approaches have been applied for a different line of genetic exploration on cannabis that described the sequence of Δ9-tetrahydrocannabinolic acid-synthase (THCAs), which is the enzyme behind the production THC from the cannabigerolic acid (CBGA) precursor.24 Indeed, the chemical production of THC has many limitations since the legal regulation of C. sativa cultivation is restricted in the majority of countries, the low yield of the asymmetric chemical synthesis of THC, and the difficult extraction of THCA. Fortunately, CBGA is easy to synthesize25 and THCA is naturally decarboxylated by simple heating or during storage.24 Therefore, a biotechnological approach could be a suitable alternative. Notably, the public server Robetta has been very efficient in protein structure prediction from the amino acid sequence. It has continuously demonstrated its reliable performance and accuracy in the CASP (Critical Assessment of Structure Prediction tests). It has been frequently evaluated by CAMEO (Continuous Automated Model EvaluatOn) since 2014.26 Moreover, the CASP10 experiment suggests that Rosetta Comparative Modeling (RosettaCM) produces models with more accurate side chain and backbone conformations compared to other methods especially when the sequence identity with the models is greater than 15% and the reference crystal structure is available. Furthermore, The complete coding region of the THCAS gene has been sequenced from DNA extracted from the resin of Moroccan cannabis.27 As of today, THCAS was only expressed in low amounts either in hairy roots, insect cells (Spodopterafrugiperda), or yeasts like Pichia pastoris (Komagataella), and Saccharomyces cerevisiae.25,28,29 Luckily in 2015, bioconversion of Pichia pastoris yeast cells led to the production of 1 mM of THCA (0.36g THCA l-1).14,30 However, cloning of THCAS gene hasn’t been yet performed in Morocco.\n\nIn order to produce the active ingredient THC in vitro, we aim to clone the THCA synthase gene extracted from Moroccan cannabis to get the recombinant enzyme, using the expression system Pichia Pastoris. We have covered in this present study the first part of our work; we isolated the THCA synthase gene from the leaves of the Moroccan variety C. sativa (khardala) without the native signal sequence, and then predicted the three-dimensional structure of the recombinant THCAS enzyme.\n\n\nMethods\n\nRaw C. sativa leaves of the variety “Khardala” were collected from Fez in northern Morocco (N 34.036466, W 5.017336), and sent for scientific investigation to the Genetics Analysis Institute of the Royal Gendarmerie, Rabat, Morocco.\n\nFirst, Cannabis leaves were ground by the Tissulyser II. Then, DNA was extracted by the ISOLATE II DNA Plant kit following the manufacturer’s instructions. Next, DNA was quantified using the NanoDrop 8000 spectrophotometer (ThermoFisher)31; one unit of absorbance (260 nm) was assumed to correspond to 50 ng of DNA per μl of solution.\n\nWe aligned the THCA synthase sequence isolated from Moroccan Cannabis resin, which has the GenBank accession number ((JQ437481)27 to the cDNA sequence obtained from the open reading frame (ORF) ARNm precursor of the THCAS gene, with the GenBank accession number (AB057805),32 deposited in NCBI.33\n\nWe deduced the specific primers of THCAS gene of the Cannabis Moroccan variety without the native signal sequence respecting the following conditions: (1) Specificity; (2) Compatibility; (3) Primer stability; (4) Primer length should be 15-30 bp; (5) Primer melting Temperature (Tm) of about 48 to 60°C; (6) Forward and Reverse primers’ Tm preferably in the range of 52-58°C; and (7) an optimum GC content ranging from 40 to 60%. These characteristics were tested by AmplifX34 and SnapGene35 softwares. The pairs of primers obtained were evaluated for non-specific hybridization to other regions of the genome using the Basic Local Alignment Search Tool (BLAST). The Multiplex Manager v.1.236 software was used to verify the primer-primer interaction, avoiding potential primer-dimers and secondary hairpin structures.\n\nWe used the same primers with adapter sequences for sub-cloning (Table 1). We first added the specific restriction site to the StuI enzyme to the sense primer. Then, three histidine codons, two transcription stop codons, and the restriction site specific to the KpnI enzyme to the antisense primer. We amplified the large chain ribulose-bisphosphate carboxylase (rbcL) gene (rbcL sense primer: 5′-ATGTCACCACAAACAGAGACTAAAGC-3′; rbcL antisense primer: 5′-GCAGCAGCTAGTTCCGGGCTCCA-3′) for the PCR positive control.37 The last step was to predict the three-dimensional structure of the recombinant enzyme THCAS exploiting deeplearning technology and the Rosetta Comparative Modeling (RosettaCM) method of the ROBETTA server.\n\nPCR amplification was performed following the standard protocol: preheating at 94°C for 5 minutes, followed by 35 cycles at 94°C for 1 minute, then at 55°C for 1 minute, and at 72°C for 2 minutes, with a final extension at 72°C for 10 minutes. The reactions were carried out in a BIO RAD S1000 thermal cycler.27\n\nThe amplified fragments were subjected to 1% of the agarose gel detected using ethidium bromide. Gels were visualized under UV trans-illuminator using the G:BOX gel system (Syngene).\n\nWe used two restriction enzymes for the digestion reaction; StuI (Sigma Aldrich), and KpnI (Roche Applied Science), which was successively performed in the L buffer from (Roche Applied Science). The first restriction enzyme reaction (StuI enzyme) was achieved using a final volume of 25 μl under the following conditions: 2.5 μl of 10X SuRe/Cut Buffer L, BSA final concentration of 100 μg/ml, and 1 μl of enzyme StuI 10 U/ul and 500 ng of DNA. The reaction was incubated for 1 hour at 37°C and inactivated by incubation for 20 min at 65°C. This was followed by a second restriction adding 1 ul of the enzyme KpnI 10 U/ul. The reaction was incubated for 1 h at 37°C and inactivated by incubation for 15 min at 65°C.\n\nGenetic individualization of THCAS gene without it’s signal sequence facilitated integration of the insert into the plasmid of choice; pPinkαHC. Thus, the amplified amplicon by two primers including adaptors was digested in addition to the vector using compatible restriction enzymes. The digested insert and vector were joined by the ligase enzyme. Actually, this reaction is primordial to succeed the sub-cloning using the chemo-competent E. coli Dhα bacterium, and hence, express the THC gene of interest in a host like the yeast Pichia pastoris.\n\n\nResults\n\nWe illustrated in Figure 1 the cDNA of the THCAS gene performed by SnapGene software in order to specify the main parts that made up the sequence of the gene in question. In addition, Figure 2 shows the alignment of THCAS gene, with the genbank accession number (JQ437481) against its formerly sequenced cDNA precursor’s counterpart, with the Genbank accession number (AB057805). The alignment result showed a high similarity of 98% starting from ATG star codon to the last three CAT (Histidine) codons of the THCAS gene.\n\nLegend: 5′UTR: Untranslated Transcribed Region 5′; 3′UTR: Untranslated Transcribed Region 3′.\n\nLegend: Query: C. sativa mRNA for tetrahydrocannabinolic Acid synthase precursor (AB057805), which is 1885 nucleotides long; Subject: Cannabis sativa isolate 01 tetrahydrocannabinolic acid synthase gene (JQ437481), which is 1635 nucléotides long; b) Alignement of the THCAS gene extracted from Moroccan Cannabis sativa with accession (JQ437481) marked in blue in relation to DNAc of ARNm with accession (AB057805) marked in Red using SnapGene software; c) Start codon (5′ ATG); Stop codon (3′ TAA).\n\nFigure 3 showed the different samples of harvested Moroccan C. sativa namely “Khardala” including duplicates to assess variance in the evaluation method, including both sampling and analysis. Samples are numbered from 1 to 6 including their weight measurement 100, 150, 200, 100, 50 and 50 mg, respectively. In addition, this figure showed the summary of DNA quantification, which varied between 75 ng/ul and 1279 ng/ul, with a ratio of 260/280 and 260/230 between 1 and 2.5.\n\nAll PCR primers with and without adapters amplified a sequence of 1551 nucleotides, from position 85N to 1635N of the THCAS gene. Additionally, amplification of the rbcl cannabis-specific positive control was successful, while no amplification was observed at the negative control (water). This result was repeated several times for confirmation with regards to samples and duplicates (Figure 4).\n\nLegend: g: amplification of DNA extract (THCAS gene); w: negative control amplification (water).\n\nWe added the forward and reverse primers to the Stul and Kpnl restriction enzymes, respectively. Histidine codons (ATG) and transcription stop codon (TTA) were added to the reverse primer. On the one hand, the enzymatic restriction was carried out in a successive manner and in both directions at the level of the pPinkα-HC vector. On the other hand, the same enzymatic restriction reactions were carried out for the amplicon produced with the primers with adapters (Figure 5).\n\nA) Vector named pPinkα-HC is composed of: 7898 nucleotides; α-factor: the signal sequence; MSC: multiple cloning site (Stu I, Kpn I, Nae I, Fse I or Swa I); 5′ AOX1 promoter region (alcohol oxidase promoter); CYC1 TT: Cytocrome C1 transcription termination; PUC ori: PUC origin of replication; AmpR: ampicillin resistance gene; P ADE2 HC: The ADE2 promoter region; ADE2: Open reading frame ADE2 (responsible for the synthesis of Adenine) TRP2: Gene TRP2 (responsible for the synthesis of tryptophan).\n\nB) Visualization on agarose gel of the pPinkα-HC vector after enzymatic digestion; 1) 1 Kb ladder; 2) undigested plasmid; 3) digested plasmid with the StuI enzyme; 4) digested plasmid with StuI enzyme, followed by the KpnI enzyme; 5) Digested plasmid with KpnI enzyme; 6) digested plasmid with Kpnl enzyme, followed by the Stul enzyme;\n\nC) Ppink-HC vector as illustrated by SnapGene software after enzymatic digestion.\n\nD) Result of PCR amplification: The amplified THCAS gene without signal sequence, the StuI restriction site is present at the 5′ position, the KpnI restriction site at the 3′ position, using the snapgen software;\n\nE) Revelation of THCAS gene without its signal sequence on agarose gel after the enzymatic digestion;\n\nF) THCAS gene without its signal sequence after enzymatic digestion as shown by SnapGene software.\n\nTHCAS gene of Moroccan C. sativa was sequenced with accession (JQ437481-JQ437488), knowing that THCAS gene has a size of 1635 nucleotides,32 and Its signal peptide sequence is 28 amino acids. Following the alignment of THCAS gene of Moroccan C. sativa against its cDNA precursor, we observed that the former allows an open reading frame of 1635 nucleotides, coding for a polypeptide of 545 amino acids (Figure 6). Thus, the length of the predicted mature THCAS polypeptide is 517 amino acids (Figures 7 and 8). We identified the NH2-terminal sequence as: NPRENFLKXFSKHIPNNVANPKLV, which corresponds to the nucleotide sequence 84-156 Ns of THCAS gene (GenBank accession number: JQ437481). It has 9 discrepancies with the NH2-terminal sequence of the cDNA (GenBank accession number: AB057805). RMSDs (root mean square deviation of atomic positions) for C-alpha atoms, main chain atoms, side chain atoms and all atoms between the modelled structure and the model corresponding to 1.325 Å (Figure 8).\n\n\nDiscussion\n\nUsage of C. sativa for pharmacological purposes, regardless of persistent controversy, helped reemerging this plant with historical significance in medical field. Morocco has been and still one the world’s largest producer of cannabis resin, with an estimated open air production of 38000 tons, and in door production of 760 tons, which helped gaining a net of an estimated 9 Billion dollars.41 Legislation with regards to governing use of cannabis for medical purposes in Morocco continues to evolve promptly42, which requires implication of scientists, doctors, and pharmacists in studying the healthy benefits of Cannabis. Moreover, THC has generated recently considerable interest for its new and valuable pharmacological activities, and hence, is been commercially available, but extremely expensive, which makes it difficult to be a target for detailed studies. Unfortunately, application of conventional methods to prepare THC, including organic synthesis and isolation from marijuana, were not very practical ways to provide enough pure THC.\n\nIn this study, we aimed to describe the recombinant THCAS gene cloning steps in order to exploit its recreational effect on the endocannabinoid system. Eventually, we sought to produce a recombinant enzyme with a specific and original enzymatic activity as well as a high production yield. To do so, we blasted the tetrahydrocannabinolic acid synthase (THCAS) gene isolated from the resin of Moroccan C. Sativa (JQ437481) corresponding to 1635 nucleotides against the mRNA precursor of THCAS (AB057805) Corresponds to 1885 nucleotides. Moreover, we prepared the nucleotide sequence of interest for subcloning the vector pPinkα-HC into E-Coli in order to express it in the yeast Komagataella phaffi (previously called Pichia pastoris). Ultimately, we predicted the final structure of the recombinant protein THCAS responsible for the production of the main active principle in Moroccan cannabis.\n\nThis work is the first step towards the expression of the recombinant THCAS enzyme in Komagataella phaffi yeast. We therefore hypothesized that recombinant THCA synthase would contribute to the biotechnological production of THC, once a suitable expression system is developed. since THCA is quantitatively decarboxylated to THC by heating. As a possible system, we have here developed the possibility of expressing recombinant THCAS in transgenic P. pastoris secreting THCA synthase. In order to achieve this goal, we started with the alignment of the THCAS sequence.\n\nSequence alignment helped identifying the conserved region that correspond to the position of key residues (his 114, cys176, his292, glu417, tyr484, Cys37 and Cys99), which are conserved in the THCAS of Moroccan C. sativa. These sites are essential for the enzymatic activity of THCAS, and found to be present at the same positions as previously described amino acids. THCAS protein, is a flavoprotein, since the presence of coenzyme FAD is essential for its enzyme activity.30 Besides, it covalently binds to His114 and Cys176.32 In addition, Tyr 484 being the catalytic site of the enzyme, including both His292 and Tyr417, are involved in binding to the CBGA substrate. the three-dimensional structure of the enzyme in question is generated thanks to the disulfide bond (between Cys37 and Cys99) and the six N-glycosylation sites.40 Interestingly, the alignment showed a 98% match, which implies a high quality. Therefore, it was not necessary to improve the alignment by correcting errors or eliminating gaps.\n\nThe yield of the extracted genomic DNA gave satisfactory, significant concentration, proportional to the mass of the initial sample. the purity of the extracted DNA was evaluated by the A260/230 ratio to assume the contamination level. The presence of polysaccharides and phenolic compounds, detergents and other salts might explain the A260/230 value, which is close to the ideal value of 2.31 The PCR quality suggests that the extract was pure enough for downstream applications, such as enzymatic DNA digestion and ligation. Meanwhile, a single band of approximately 1600 nucleotides was obtained after enzymatic restriction. Hence, we deduced that the choice of restriction enzymes was successful because they cut at the level of the THCAS gene of interest and recognized a restriction site composed of 6 nucleotides to maintain the same reading frame of the gene of interest. Furthermore, the three histidine codons were added to facilitate the extraction of the recombinant enzyme by HPLC chromatography.\n\nIn the first run, we performed enzymatic restriction with StuI and KpnI at the same time, because the two enzymes have 100% activity in the SuRe/Cut Buffer L. However, both enzymes lost their enzymatic activity, which implies that the presence of the two reacting enzymes initiates their activities. Consequentially, we carried out a restriction in a successive way and in both directions to ensure that the activity of each enzyme remains. Later, the same reactions were performed for the amplicon produced by the adapter primers. This result may be useful for applications like ligase reaction and molecular cloning, which means that primers design was successful. However, a sequencing step would be crucial for confirmation.\n\nTHCA synthase is identified as a monomeric enzyme. It consists of two domains (Domains I and II) divided by the FAD.40 The formation of the oxidative cyclization reaction will likely occur through intermediaries.43 Along the entire length of the THCAS enzyme protein, there are positions that interact with the CBGA substrate or the FAD cofactor. This leads us to the need for molecular cloning of the entire nucleotide sequence, as has been done previously.14,29,32 In addition to obtaining greater enzymatic activity of the recombinant enzyme. Endoglycosidase treatment yielded deglycosylated THCA synthase with higher catalytic activity than either the glycosylated form of the native enzyme or the recombinant enzyme.29\n\nFurthermore, the RMSD of the modeled structures and the corresponding reference model is 1.325 Å, reflecting the high quality of the obtained models. This result is essential for earlier applications such as molecular cloning. DeepMind’s Alphafold2 demonstrated its remarkable robustness and accuracy for the example of our enzyme of interest with this result. Each possible change in the nucleotide sequence made by the site-directed mutation during molecular cloning can be tested in silico before carrying it out at the laboratory level by modeling in X-ray crystallography and cryo-electron microscopy and generating precise models of complexes. protein-protein and saved considerable time.26\n\nAdditionally, The Rosetta Comparative Modeling (RosettaCM) experiment produced models with more accurate side chain and backbone conformations than other methods when the sequence identity with the models is greater than 15% and the crystal structure of the model is arranged. Other works have been executed in this sense by the same THCAS gene of Moroccan origin using conventional techniques such as Modeling.44 Remarkably, the same modeling accuracy was approximately obtained.\n\nUnfortunately, little is still known about THCAS in Cannabis, which constitutes a valuable unexploited treasure. However, artificial intelligence is being applied by pharmaceutical companies to discover drugs and design new optimized compounds in order to accelerate the identification of promising drug candidates for specific indications. Hence, we applied this technique on the THCAS of Cannabis sativa for the first time in Morocco. The use of the 3VTE reference structure helped improving both the number of aligned residues and the quality of the superposition, evaluated by the root mean square deviation of atomic position (RMSD) calculation. Furthermore, the modeled protein was superimposed to the reference structure to assess the predicted three-dimensional structure quality, which showed a good conservation of the model’s global folding. This would foster a better understand of the crucial conformation at the active principle level, which could explain the variation in the rate of THCA in the simulated model by bioinformatics. We ultimately aim to generate a molecular cloning protocol of THCAS gene of interest and hence, predict the most relevant recombinant enzyme for gene expression in yeast. Therefore, we will be saving time, energy and resources in laboratory work. However, this method remains incomplete to judge the reliability of the recombinant enzyme. It is advantageous to go through in vitro and in vivo experiments before any human drug testing.\n\n\nConclusion\n\nThis study demonstrated a great similarity between the THCAS gene of Moroccan Cannabis variety and the formerly reported cDNA precursor gene reference. We isolated the nucleotide sequence of the THCAS gene using targeting specifics primers in order to express this gene in an appropriate host. Thus, we were able to predict for the first time, the structure of the recombinant THCAS enzyme using in silico, in vitro, and artificial intelligence tools. These results are essential to successfully clone and express the THCA recombinant enzyme in a eukaryotic host such as the yeast Pichia pastoris.\n\n\nData availability\n\nThe THCAS sequences with a complete open reading frame sequenced from Moroccan cannabis resin are available under these Genbank accession numbers27:\n\nGenBank: Cannabis sativa isolate 01 tetrahydrocannabinolic acid synthase gene, partial cds. Accession number: JQ437481.\n\nGenBank: Cannabis sativa isolate 02 tetrahydrocannabinolic acid synthase gene, partial cds. Accession number: JQ437482.\n\nGenBank: Cannabis sativa isolate 03 tetrahydrocannabinolic acid synthase gene, partial cds. Accession number: JQ437483.\n\nGenBank: Cannabis sativa isolate 04 tetrahydrocannabinolic acid synthase gene, partial cds. Accession number: JQ437484.\n\nGenBank: Cannabis sativa isolate 05 tetrahydrocannabinolic acid synthase gene, partial cds. Accession number: JQ437485.\n\nGenBank: Cannabis sativa isolate 06 tetrahydrocannabinolic acid synthase gene, partial cds. Accession number: JQ437486.\n\nGenBank: Cannabis sativa isolate 07 tetrahydrocannabinolic acid synthase gene, partial cds. Accession number: JQ437487.\n\nGenBank: Cannabis sativa isolate 08 tetrahydrocannabinolic acid synthase gene, partial cds. Accession number: JQ437488.\n\nDNAc of theTHCAS precursor ARNm in C. sativa sequenced in Japan, used for comparative bioninformatics analysis is available under the GenBank accession number32:\n\nGenBank: Cannabis sativa mRNA for tetrahydrocannabinolic acid synthase precursor, complete cds. Accession number: AB057805.",
"appendix": "Acknowledgments\n\nI would like to express my sincere thanks to all co-authors. I am highly indebted to my supervisors for their guidance and constant mentoring as well as for providing necessary information regarding the study and also for their support in completing the project.\n\n\nReferences\n\nBen Amar M: Cannabinoids in Medicine: A Review of Their Therapeutic Potential. J. Ethnopharmacol. 2006; 105: 1–25. PubMed Abstract | Publisher Full Text\n\nChouvy P-A: Production de Cannabis et de Haschich Au Maroc: Contexte et Enjeux. L’Espace Polit. 2008. Publisher Full Text\n\nRobson P: Human Studies of Cannabinoids and Medicinal Cannabis. Handb. Exp. Pharmacol. 2005; 168: 719–756. Publisher Full Text\n\nPertwee RG: Cannabinoid Pharmacology: The First 66 Years: Cannabinoid Pharmacology. Br. J. Pharmacol. 2009; 147 Suppl 1: S163–S171. PubMed Abstract | Publisher Full Text\n\nAyakar SR, Pawar SV, Hallam SJ, et al.: Metabolic Engineering of e. Coli for the Biosynthesis of Cannabinoid Products.2019.\n\nMendez M, Joseph N, Michael B, et al.: Compositions and Methods for Using Genetically Modified Enzymes.2019.\n\nGajewski J, Pavlovic R, Fischer M, et al.: Engineering Fungal de Novo Fatty Acid Synthesis for Short Chain Fatty Acid Production. Nat. Commun. 2017; 8: 14650. PubMed Abstract | Publisher Full Text\n\nTan Z, Clomburg JM, Gonzalez R: Synthetic Pathway for the Production of Olivetolic Acid in Escherichia Coli. ACS Synth. Biol. 2018; 7: 1886–1896. PubMed Abstract | Publisher Full Text\n\nKayser O, Stehle F-O: Biotechnological Production of Cannabinoids.2020.\n\nSchmitt P, Taboada A: Production of Plant-Based Active Substances (e.g., Cannabinoids) by Recombinant Microorganisms.2020.\n\nMelis A, Betterle N, Martinez HAD: Production of Cannabinoids Using Genetically Engineered Photosynthetic Microorganisms.2020.\n\nLaban A: Cannabinoid Production in Algae.2019.\n\nPoulos JL, Farnia AN: Production of Cannabinoids in Yeast.2016.\n\nZirpel B, Stehle F, Kayser O: Production of Δ9-Tetrahydrocannabinolic Acid from Cannabigerolic Acid by Whole Cells of Pichia (Komagataella) Pastoris Expressing Δ9-Tetrahydrocannabinolic Acid Synthase from Cannabis sativa L. Biotechnol. Lett. 2015; 37: 1869–1875. PubMed Abstract | Publisher Full Text\n\nZirpel B, Degenhardt F, Martin C, et al.: Engineering Yeasts as Platform Organisms for Cannabinoid Biosynthesis. J. Biotechnol. 2017; 259: 204–212. PubMed Abstract | Publisher Full Text\n\nLuo X, Reiter MA, d’Espaux L, et al.: Complete Biosynthesis of Cannabinoids and Their Unnatural Analogues in Yeast. Nature. 2019; 567: 123–126. Publisher Full Text\n\nMookerjee S, Campbell JA, Wiltshire ZD, et al.: Method and Cell Line for Production of Phytocannabinoids and Phytocannabinoid Analogues in Yeast.2022.\n\nZirpel B, Degenhardt F, Zammarelli C, et al.: Optimization of Δ 9 -Tetrahydrocannabinolic Acid Synthase Production in Komagataella Phaffii via Post-Translational Bottleneck Identification. J. Biotechnol. 2018; 272-273: 40–47. PubMed Abstract | Publisher Full Text\n\nZirpel B, Kayser O, Stehle F: Elucidation of Structure-Function Relationship of THCA and CBDA Synthase from Cannabis Sativa L. J. Biotechnol. 2018; 284: 17–26. Publisher Full Text\n\nBeardslee TA: Biosynthetic Cannabinoid Production in Engineered Microorganisms.2020.\n\nGülck T, Booth JK, Carvalho Â, et al.: Synthetic Biology of Cannabinoids and Cannabinoid Glucosides in Nicotiana Benthamiana and Saccharomyces Cerevisiae. J. Nat. Prod. 2020; 83: 2877–2893. Publisher Full Text\n\nSayre RT, Gonçalves EC, Zidenga T: High Level in vivo Biosynthesis and Isolation OfWater Soluble Cannabinoids in Stably Transformed Plant Systems.2019.\n\nGeissler M, Volk J, Stehle F, et al.: Subcellular Localization Defines Modification and Production of Δ9-Tetrahydrocannabinolic Acid Synthase in Transiently Transformed Nicotiana Benthamiana. Biotechnol. Lett. 2018; 40: 981–987. PubMed Abstract | Publisher Full Text\n\nYamauchi T, Shoyama Y, Aramaki H, et al.: Tetrahydrocannabinolic Acid, a Genuine Substance of Tetrahydrocannabinol. Chem. Pharm. Bull. 1967; 15: 1075–1076. Publisher Full Text\n\nSirikantaramas S, Morimoto S, Shoyama Y, et al.: The Gene Controlling Marijuana Psychoactivity: Molecular Cloning and Heterologous Expression of Δ1-Tetrahydrocannabinolic Acid Synthase from Cannabis Sativa L. J. Biol. Chem. 279: 39767–39774. PubMed Abstract\n\nBaek M, DiMaio F, Anishchenko I, et al.: Accurate Prediction of Protein Structures and Interactions Using a Three-Track Neural Network. Science. 2021; 373: 871–876. PubMed Abstract | Publisher Full Text\n\nEl Alaoui MA, Melloul M, Alaoui Amine S, et al.: Extraction of High Quality DNA from Seized Moroccan Cannabis Resin (Hashish). PLoS One. 2013; 8: e74714. PubMed Abstract | Publisher Full Text\n\nBadrana F, Mokhtari A, Safini N, et al.: Systematic Review and Meta-Analysis for the Biotechnological Production of THC in Morocco. E3S Web Conf. 2021; 319: 02012. Publisher Full Text\n\ntaura Production of D1-Tetrahydrocannabinolic Acid by the Biosynthetic Enzyme Secreted from Transgenic Pichia Pastoris.2007.\n\nBlatt-Janmaat K, Qu Y: The Biochemistry of Phytocannabinoids and Metabolic Engineering of Their Production in Heterologous Systems. Int. J. Mol. Sci. 2021; 22: 2454. PubMed Abstract | Publisher Full Text\n\nNanoDrop Technical Support Bulletin T009.2007.\n\nSirikantaramas S, Morimoto S, Shoyama Y, et al.: The Gene Controlling Marijuana Psychoactivity. J. Biol. Chem. 2004; 279: 39767–39774. PubMed Abstract | Publisher Full Text\n\nNCBI:2022.Reference Source\n\nNicolas J: AmplifX.2004.\n\nGlick B: Snapgene.2017.\n\nHolleley; Geerts Multiplex Manager v.1.2. 2009.\n\nKojoma M, Seki H, Yoshida S, et al.: DNA Polymorphisms in the Tetrahydrocannabinolic Acid (THCA) Synthase Gene in “Drug-Type” and “Fiber-Type” Cannabis sativa L. Forensic Sci. Int. 2006; 159: 132–140. PubMed Abstract | Publisher Full Text\n\nDeLano WL: PYMOL. Schrödinger Inc.;2010.\n\nShoyama Y, Tamada T, Kurihara K, et al.: Structure and Function of ∆1-Tetrahydrocannabinolic Acid (THCA) Synthase, the Enzyme Controlling the Psychoactivity of Cannabis sativa. J. Mol. Biol. 2012; 423: 96–105. PubMed Abstract | Publisher Full Text\n\nShoyama Y, Tamada T, Kurihara K, et al.: Structure and Function of ∆1-Tetrahydrocannabinolic Acid (THCA) Synthase, the Enzyme Controlling the Psychoactivity of Cannabis sativa. J. Mol. Biol. 2012; 423: 96–105. PubMed Abstract | Publisher Full Text\n\nUNODC: World Drug Report 2016. Vienna, New York:United Nations Office on Drugs and Crime;2016.\n\nLoi N° 13-21.2021.\n\nTaura F, Morimoto S, Shoyama Y, et al.: First Direct Evidence for the Mechanism of.DELTA.1-Tetrahydrocannabinolic Acid Biosynthesis. J. Am. Chem. Soc. 1995; 117: 9766–9767. Publisher Full Text\n\nEl Alaoui MA, Ibrahimi A, Semlali O, et al.: Affinity Comparison of Different THCA Synthase to CBGA Using Modeling Computational Approaches. Bioinformation. 2014; 10: 33–38. PubMed Abstract | Publisher Full Text"
}
|
[
{
"id": "149670",
"date": "10 Oct 2022",
"name": "Supaart Sirikantaramas",
"expertise": [
"Reviewer Expertise Plant biochemistry and molecular biology"
],
"suggestion": "Not Approved",
"report": "Not Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nBadrana et al. reported a nucleotide sequence of the gene encoding THCA synthase from Moroccan C. sativa, a cloning of this gene into yeast expression vector, and molecular modeling of the enzyme. In my opinion, this manuscript is not in the form of research article. It reports many preliminary steps like quantification of DNA using nanodrop, primer designing, agarose gel electrophoresis, and cloning. To me, it is is a brief report.\nThe objectives of this manuscript are not clear. The authors mentioned that the cloning of THCAS gene in Moroccan cultivar has not been studied but with this reason it is not interesting or reasonable enough to perform the study. If the Moroccan cultivar can produce THCA, then definitely THCAS sequence would be highly identical. The authors aimed to clone and express in Pichia which have already been done1. On top of that, the authors have not reported the production of the recombinant enzyme, just reported the cloning step which is not necessary for an research article (this is a part of methods, not results to be shown)\nThe authors predicted the 3D structure of the recombinant protein. This sounds strange to me and not reasonable. In fact the nucleotide sequence of the Moroccan cultivar show 98% identity, not similarity as mentioned by the authors. So, I expected not to see many differences between the reported crystal structure of THCAS.\nThe authors mentioned that little is known on THCAS in Cannabis, of which I disagreed. The catalytic mechanisms has been reported and the crystal structure has been published2. Many biotechnological applications using THCAS genes have been extensively reported3.\nOverall, I appreciated the time invested on this work by the authors but, sorry to say, I think this manuscript is a premature stage of research. Much more works are needed to be done with a clear and novel objectives.\n\nOne last thing, in many places the authors used ARNm and DNAc which I suspected to be read as mRNA and cDNA in the figure legends. English needs to be checked as well.\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? No",
"responses": [
{
"c_id": "8914",
"date": "21 Oct 2022",
"name": "Fadwa Badrana",
"role": "Author Response",
"response": "Dear reviewer, Thank you for your interest in our paper “In silico and in vitro analysis of THCA synthase gene in Moroccan Cannabis Sativa, L”. As recommended, we have addressed your comments point by point and we revised our manuscript accordingly. I have taken note of your comments, but please allow me to state the reasons why I disagree. Comment of the reviewer: “Badrana et al. reported a nucleotide sequence of the gene encoding THCA synthase from Moroccan C. sativa, a cloning of this gene into yeast expression vector, and molecular modeling of the enzyme.” Response of the authors: The sequencing of the THCAS gene from the cannabis resigne of Moroccan origin was carried out in 2014. Therefore, it was thought to individualize the THCAS gene without the signal sequence for its colonage the yeast expression vector. Thus the prediction of its three-dimensional structure of the enzyme with the new tool of artificial intelligence (Beak, 2021). Comment of the reviewer: “In my opinion, this manuscript is not in the form of research article. It reports many preliminary steps like quantification of DNA using nanodrop, primer designing, agarose gel electrophoresis, and cloning. To me, it is is a brief report.” Response of the authors: A paper or article is defined as a document describing, reviewing or discussing results, observations, hypotheses, theories, methods, and other outputs of academic and engineering research. here we described and discussed our results and the importance and utility of our work which is molecular cloning and prediction via artificial intelligence. This article doesn't contain a fatal error in design or presentation of data (e.g., inappropriate methodology or analyses), lacks originality or little new information to the current literature, has little significance or relevance to other institutions, doesn't answer the research question. The objective of this research is to clone the THCAS gene for the first time in Morocco from Moroccan cannabis. Comment of the reviewer: “The objectives of this manuscript are not clear. The authors mentioned that the cloning of THCAS gene in Moroccan cultivar has not been studied but with this reason it is not interesting or reasonable enough to perform the study. If the Moroccan cultivar can produce THCA, then definitely THCAS sequence would be highly identical. The authors aimed to clone and express in Pichia which have already been done1. On top of that, the authors have not reported the production of the recombinant enzyme, just reported the cloning step which is not necessary for an research article (this is a part of methods, not results to be shown)” Response of the authors: the objective of our work is quite clear. First, the cloning of the THCAS gene has never been carried out in Morocco: this research is a first. In addition, Morocco’s ranking as the world’s leading producer and exporter of cannabis (UNODC, 2022), the reputation of these Moroccan varieties (climate and atmosphere) among European consumers, on the one hand. the legalization of medical cannabis in Morocco on the other hand makes the study of this variety in question necessary in particular the enzyme THCAS responsible for the formation of the only psychoactive molecule THC. The Moroccan variety stimulates in-depth biochemical studies, Genetic and structural among which our preliminary study for the production of recombinant enzymes and prediction of its three-dimensional structure without going through crystallization as previously reported. The expression pichia gave insufficient rates for industrial pharmaceutical production. For this reason, research is still underway to increase recombinant protein yield. This is what we hope to achieve through this first part of the research. Comment of the reviewer: “The authors predicted the 3D structure of the recombinant protein. This sounds strange to me and not reasonable. In fact the nucleotide sequence of the Moroccan cultivar show 98% identity, not similarity as mentioned by the authors. So, I expected not to see many differences between the reported crystal structure of THCAS.” Response of the authors: 98% identity may be insufficient to say similar, if 2% difference affects catalytic and substrate fixation sites. This is not the case for us, but on the other hand can influence the production rate of active ingredient. So to understand, you had to go through the precise structural prediction of deep learning. This method can later help us to imagine point mutations that can increase enzyme activity. But for now, we have compared this new technique with conventional techniques. Indeed, the quality of the structure encourages us to use it and save time, precision and blow. Comment of the reviewer: “The authors mentioned that little is known on THCAS in Cannabis, of which I disagreed. The catalytic mechanisms has been reported and the crystal structure has been published. Many biotechnological applications using THCAS genes have been extensively reported3. ” Response of the authors: I agreed. But, why the enzyme activity differs between varieties, this is what we want to understand at the molecular level (DNA and protein). Now we know that climate and irrigation and position in relation to the equatorial line influence the growth of the plant and the rate of production of cannabinoids. However, it is not yet known how these parameters translate at the molecular level. Comment of the reviewer: “Overall, I appreciated the time invested on this work by the authors but, sorry to say, I think this manuscript is a premature stage of research. Much more works are needed to be done with a clear and novel objectives.” Response of the authors: Multiscale interdisciplinary approaches have been integrated, combining bioinformatics, modelling and artificial intelligence in silico and in vitro experiments. To highlight the potential of these processes on the Moroccan variety of cannabis Sativa Comment of the reviewer: ”One last thing, in many places the authors used ARNm and DNAc which I suspected to be read as mRNA and cDNA in the figure legends. English needs to be checked as well.” Response of the authors: A paper doesn't meet writing standards (e.g., lacks organization or clarity, has inadequate description and interpretation of results), and doesn't meet the journal's requirements for submission of articles. However ARNm and DNAc must be changed to mRNA and cDNA. All the changes we did are underlined in the new version of the manuscript. We sincerely hope these explanation and modifications will satisfy you. References: Baek M, DiMaio F, Anishchenko I, Dauparas J, Ovchinnikov S, Lee GR, Wang J, Cong Q, Kinch LN, Schaeffer RD, Millán C, Park H, Adams C, Glassman CR, DeGiovanni A, Pereira JH, Rodrigues AV, van Dijk AA, Ebrecht AC, Opperman DJ, Sagmeister T, Buhlheller C, Pavkov-Keller T, Rathinaswamy MK, Dalwadi U, Yip CK, Burke JE, Garcia KC, Grishin NV, Adams PD, Read RJ, Baker D. Accurate prediction of protein structures and interactions using a three-track neural network. Science. 2021 Aug 20;373(6557):871-876. doi: 10.1126/science.abj8754. Epub 2021 Jul 15. PMID: 34282049; PMCID: PMC7612213. UNODC :United Nations Office on Drugs and Crime;. Nigeria Cannabis Survey. 2022."
}
]
},
{
"id": "153183",
"date": "07 Nov 2022",
"name": "Noureddine Hamamouch",
"expertise": [
"Reviewer Expertise Plant Molecular Biology and Biotechnology."
],
"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\nEditorial Note from F1000Research - 22nd December 2022: It has come to our attention that Dr Noureddine Hamamouch is the PhD supervisor for Bouchra Chaouni. The conflict of interest statement has been updated to reflect this, and Dr Noureddine will not be invited to review further versions of this article.\nTitle : 'sativa' (with small s) not 'Sativa'.\nPage 1:\nIn title: in vitro analysis, I am not what the authors meant by in vitro analysis of the gene, the author claimed that the gene was “cloned”, but not further analysis was conducted on the gene products.\n\nThe last sentence in the abstract, the authors said that “basic molecular biology for the THCAS gene expression remains largely unstudied”. This is true, however, expression analysis was not conducted in the study.\n\nFirst sentence in “Methods section”: “ in this paper, we explored…\" - this is the objective of the study and should not be in the “Methods” section.\n\nIn the “Methods section” the authors said that they used conventional experiments.” They need to specify exactly what methods were used. Also in the same section, the authors said that they used markers, what kind of markers? Do you mean “Primers”?\n\nIn the results section: the authors claimed that the main finding was, “high sequence similarity with THCAS and the mRNA precursor of the same gene as previously reported”. This is not clear, how did they found similarity if the gene they claimed cloned was not sequenced?\n\nPage 2:\nThe two Arabic words used in the “introduction” should be removed.\n\nIn the “Introduction, line 11, \"…has been attempted by different microorganisms\", must be changed to, \"...has been attempted in different microorganisms\".\n\nThe heterologous production of cannabinoids in different organisms, need to be developed further, it is weak in its current form. The authors also need to explain the challenges associated with heterologous expression of THCAS gene.\n\nThe author said in line (under Introduction) that the THCAS gene has been expressed in yeast cells (references 25, 28, and 29), and said the objective of their study was to express this gene in yeast cells, if it has been expressed before why the authors want to express it again, were there problems with its expression before? What kind of solutions the authors are proposing?\n\nThe authors said that the objective of the study was “to clone THCA synthase gene from Moroccan cannabis to get the recombinant enzyme”. The authors did not show neither par PCR, or restriction enzyme digestion, of sequencing that they have cloned the gene, nor did they show that they got the recombinant enzyme. What they claim (last sentence in the Introduction section), \"we isolated the THCA synthase gene from the leaves of the Moroccan variety C. sativa, without the native signal sequence and then predicted the three dimensional structure of the recombinant THCAS enzyme\". This a serious problem, because the authors need to verify that they have indeed isolated the gene of interest, and they need to do that by sequencing, without sequencing there is not proof that they gene cloned is indeed the THCA. Moreover, if no sequencing was conducted, what sequence did the author use to predict the three dimensional structure of the gene. The methods section: some important details need to be added:\n- Was the plant harvested identified by a plant taxonomist? - How was the plant materials send for analysis? - Was the plant material used in DNA fresh or dried? And how much was the starting material?\n\nThe authors need to explain when the sequence of the signal peptide was not include in the design of primers, and what is the objective for including 3 HIS codon?\nPage 5:\n\nUnder DNA extraction and quantification, the is an error in the quantities of DNA used: 50 mg is repeated twice.\n\nPlease combine the sections: 1) DNA extraction and quantification 2) PCR amplification and 3) Restriction enzyme digestion\nPage7;\nWhy was the RUBISCO gene considered as a positive control? It should not, the positive control should be THCAS gene and not RUBISCO.\nUnder Discussion:\nFigures 5, 6, 7, and 8 are results and should therefore not be in the discussion section.\n\nFigure 5 : Insert not INSER / 1 Kb Ladder not LADDR.\n\nFigure 5: please use a better photo, that one is not clear.\n\nFigure 5 “INSERT” - It is not Insert it is a PCR product after digestion, the author did not show that they have actually inserted the gene in a plasmid, so it is just a PCR product? Right?\n\nFigure 5: the 1KB ladder is not well separated on a gel, and please indicate the sizes of the 1 Kb ladder.\n\nThe authors did actually insert the gene in the plasmid, they only amplified the gene by PCR and prepared it for cloning, the cloning steps have not been described.\nConclusion:\nThe authors claimed that their study demonstrated a great similarity between the THCAS gene of Cannabis Moroccan variety and the reported cDNA precursor gene. However, no sequencing has been conducted to support their claim.\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Partly\n\nAre sufficient details of methods and analysis provided to allow replication by others? Partly\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nNot applicable\n\nAre all the source data underlying the results available to ensure full reproducibility? Partly\n\nAre the conclusions drawn adequately supported by the results? No",
"responses": [
{
"c_id": "9046",
"date": "15 Dec 2022",
"name": "Fadwa Badrana",
"role": "Author Response",
"response": "Title: 'sativa' (with small s) not 'Sativa'. Page 1: In title: in vitro analysis, I am not what the authors meant by in vitroanalysis of the gene, the author claimed that the gene was “cloned”, but not further analysis was conducted on the gene products. The last sentence in the abstract, the authors said that “basic molecular biology for the THCAS gene expression remains largely unstudied”. This is true, however, expression analysis was not conducted in the study. First sentence in “Methods section”: “ in this paper, we explored…\" - this is the objective of the study and should not be in the “Methods” section. In the “Methods section” the authors said that they used conventional experiments.” They need to specify exactly what methods were used. Also in the same section, the authors said that they used markers, what kind of markers? Do you mean “Primers”? In the results section: the authors claimed that the main finding was, “high sequence similarity with THCAS and the mRNA precursor of the same gene as previously reported”. This is not clear, how did they found similarity if the gene they claimed cloned was not sequenced? Answer page 1: In vitro analysis, qualifies a biological process observed and or studied in the laboratory, under artificial conditions, as opposed to in vivo. In this study, we cloned the THCAS gene from Moroccan cannabis into an expression vector PpinkαHC, to transform the E. coli bacteria, for future expression in the yeast Pichia pasteri. The first steps of the molecular cloning were performed in the laboratory. We explored the protocol of molecular cloning of the recombinant THCAS gene for the Moroccan variety Cannabis sativa. We also described the conventional experiments performed in vitro. we individualized the THCAS gene without its own sequence signaled by our primers. transforming the bacteria E. coli DHɑ chemocompetent. These are the steps we have been able to achieve up to now Markers define primers and conventional methods are PCR, primer design and use of restriction enzymes as well as ligation and transformation of E. Coli DHɑ bacteria. A THCAS gene DNA from cannabis of Moroccan origin was compared with previously reported precursor mRNA (cDNA) of the same gene. Content changes are included in the article. Page 2: The two Arabic words used in the “introduction” should be removed. In the “Introduction, line 11, \"…has been attempted by different microorganisms\", must be changed to, \"...has been attempted in different microorganisms\". The heterologous production of cannabinoids in different organisms, need to be developed further, it is weak in its current form. The authors also need to explain the challenges associated with heterologous expression of THCAS gene. The author said in line (under Introduction) that the THCAS gene has been expressed in yeast cells (references 25, 28, and 29), and said the objective of their study was to express this gene in yeast cells, if it has been expressed before why the authors want to express it again, were there problems with its expression before? What kind of solutions the authors are proposing? The authors said that the objective of the study was “to clone THCA synthase gene from Moroccan cannabis to get the recombinant enzyme”. The authors did not show neither par PCR, or restriction enzyme digestion, of sequencing that they have cloned the gene, nor did they show that they got the recombinant enzyme. What they claim (last sentence in the Introduction section), \"we isolated the THCA synthase gene from the leaves of the Moroccan variety C. sativa, without the native signal sequence and then predicted the three dimensional structure of the recombinant THCAS enzyme\". This a serious problem, because the authors need to verify that they have indeed isolated the gene of interest, and they need to do that by sequencing, without sequencing there is not proof that they gene cloned is indeed the THCA. Moreover, if no sequencing was conducted, what sequence did the author use to predict the three dimensional structure of the gene. The methods section: some important details need to be added - Was the plant harvested identified by a plant taxonomist? - How was the plant materials send for analysis? - Was the plant material used in DNA fresh or dried? And how much was the starting material? The authors need to explain when the sequence of the signal peptide was not include in the design of primers, and what is the objective for including 3 HIS codon? Answer page 2: E. coli was not an appropriate host for the expression of the recombinant THCAS enzyme (previously reported). Experiments with THCA production in methylotrophic yeast have shown promising results using a Pichia pastoris expression system. In addition, recombinant THCAS purified from P. pastoris had a much higher level of activity than native THCAS purified from C. Sativa and recombinant THCAS produced by insect cells. However, until now, the expression taut of recombinant enzymes remains insufficient for an industrial expression. This experience was completed just prior to the COVID pandemic lockdown. So the feasibility of the sequence technique was not feasible. The gene already contains 3 codon his in position 3', we wanted to add the 3 HIS to simplify the purification step with chromatography after expression of the gene to the yeast Content changes are included in the article. Page 5: Under DNA extraction and quantification, the is an error in the quantities of DNA used: 50 mg is repeated twice. Please combine the sections: 1) DNA extraction and quantification 2) PCR amplification and 3) Restriction enzyme digestion Answer page 5: 50 mg is in double determination because it is a small amount. Content changes are included in the article. Page 7; Why was the RUBISCO gene considered as a positive control? It should not, the positive control should be THCAS gene and not RUBISCO. Under Discussion: Figures 5, 6, 7, and 8 are results and should therefore not be in the discussion section. Figure 5 : Insert not INSER / 1 Kb Ladder not LADDR. Figure 5: please use a better photo, that one is not clear. Figure 5 “INSERT” - It is not Insert it is a PCR product after digestion, the author did not show that they have actually inserted the gene in a plasmid, so it is just a PCR product? Right? Figure 5: the 1KB ladder is not well separated on a gel, and please indicate the sizes of the 1 Kb ladder. The authors did actually insert the gene in the plasmid, they only amplified the gene by PCR and prepared it for cloning, the cloning steps have not been described. Answer page 7: The RUBISCO gene (large chain ribulose-bisphosphate carboxylase gene) is a home gene. It is a positive control of PCR. Content changes are included in the article. Conclusion: The authors claimed that their study demonstrated a great similarity between the THCAS gene of Cannabis Moroccan variety and the reported cDNA precursor gene. However, no sequencing has been conducted to support their claim. Answer conclusion This experience was completed just prior to the COVID-19 pandemic lockdown. So the feasibility of the sequence technique was not feasible."
},
{
"c_id": "9107",
"date": "15 Dec 2022",
"name": "Fadwa Badrana",
"role": "Author Response",
"response": "Title: 'sativa' (with small s) not 'Sativa'. Page 1: In title: in vitro analysis, I am not what the authors meant by in vitro analysis of the gene, the author claimed that the gene was “cloned”, but not further analysis was conducted on the gene products. The last sentence in the abstract, the authors said that “basic molecular biology for the THCAS gene expression remains largely unstudied”. This is true, however, expression analysis was not conducted in the study. First sentence in “Methods section”: “ in this paper, we explored…\" - this is the objective of the study and should not be in the “Methods” section. In the “Methods section” the authors said that they used conventional experiments.” They need to specify exactly what methods were used. Also in the same section, the authors said that they used markers, what kind of markers? Do you mean “Primers”? In the results section: the authors claimed that the main finding was, “high sequence similarity with THCAS and the mRNA precursor of the same gene as previously reported”. This is not clear, how did they found similarity if the gene they claimed cloned was not sequenced? Answer page 1: In vitro analysis, qualifies a biological process observed and or studied in the laboratory, under artificial conditions, as opposed to in vivo. In this study, we cloned the THCAS gene from Moroccan cannabis into an expression vector PpinkαHC, to transform the E. coli bacteria, for future expression in the yeast Pichia pasteri. The first steps of the molecular cloning were performed in the laboratory. We explored the protocol of molecular cloning of the recombinant THCAS gene for the Moroccan variety Cannabis sativa. We also described the conventional experiments performed in vitro. we individualized the THCAS gene without its own sequence signalled by our primers. transforming the bacteria E. coli DHɑ chemocompetent. These are the steps we have been able to achieve up to now Markers define primers and conventional methods are PCR, primer design and use of restriction enzymes as well as ligation and transformation of E. coli DHɑ bacteria. A THCAS gene DNA from cannabis of Moroccan origin was compared with previously reported precursor mRNA (cDNA) of the same gene. Content changes are included in the article. Page 2: The two Arabic words used in the “introduction” should be removed. In the “Introduction, line 11, \"…has been attempted by different microorganisms\", must be changed to, \"...has been attempted in different microorganisms\". The heterologous production of cannabinoids in different organisms, need to be developed further, it is weak in its current form. The authors also need to explain the challenges associated with heterologous expression of THCAS gene. The author said in line (under Introduction) that the THCAS gene has been expressed in yeast cells (references 25, 28, and 29), and said the objective of their study was to express this gene in yeast cells, if it has been expressed before why the authors want to express it again, were there problems with its expression before? What kind of solutions the authors are proposing? The authors said that the objective of the study was “to clone THCA synthase gene from Moroccan cannabis to get the recombinant enzyme”. The authors did not show neither par PCR, or restriction enzyme digestion, of sequencing that they have cloned the gene, nor did they show that they got the recombinant enzyme. What they claim (last sentence in the Introduction section), \"we isolated the THCA synthase gene from the leaves of the Moroccan variety C. sativa, without the native signal sequence and then predicted the three dimensional structure of the recombinant THCAS enzyme\". This a serious problem, because the authors need to verify that they have indeed isolated the gene of interest, and they need to do that by sequencing, without sequencing there is not proof that they gene cloned is indeed the THCA. Moreover, if no sequencing was conducted, what sequence did the author use to predict the three dimensional structure of the gene. The methods section: some important details need to be added: - Was the plant harvested identified by a plant taxonomist? - How was the plant materials send for analysis? - Was the plant material used in DNA fresh or dried? And how much was the starting material? The authors need to explain when the sequence of the signal peptide was not include in the design of primers, and what is the objective for including 3 HIS codon? Answer page 2: E. coli was not an appropriate host for the expression of the recombinant THCAS enzyme (previously reported). Experiments with THCA production in methylotrophic yeast have shown promising results using a Pichia pastoris expression system. In addition, recombinant THCAS purified from P. pastoris had a much higher level of activity than native THCAS purified from C. Sativa and recombinant THCAS produced by insect cells. However, until now, the expression taut of recombinant enzymes remains insufficient for an industrial expression. This experience was completed just prior to the COVID pandemic lockdown. So the feasibility of the sequence technique was not feasible. The gene already contains 3 codon HIS in position 3', we wanted to add the 3 HIS to simplify the purification step with chromatography after expression of the gene to the yeast Content changes are included in the article. Page 5: Under DNA extraction and quantification, the is an error in the quantities of DNA used: 50 mg is repeated twice. Please combine the sections: 1) DNA extraction and quantification 2) PCR amplification and 3) Restriction enzyme digestion Answer page 5: 50 mg is in double determination because it is a small amount. Content changes are included in the article. Page 7; Why was the RUBISCO gene considered as a positive control? It should not, the positive control should be THCAS gene and not RUBISCO. Under Discussion: Figures 5, 6, 7, and 8 are results and should therefore not be in the discussion section. Figure 5 : Insert not INSER / 1 Kb Ladder not LADDR. Figure 5: please use a better photo, that one is not clear. Figure 5 “INSERT” - It is not Insert it is a PCR product after digestion, the author did not show that they have actually inserted the gene in a plasmid, so it is just a PCR product? Right? Figure 5: the 1KB ladder is not well separated on a gel, and please indicate the sizes of the 1 Kb ladder. The authors did actually insert the gene in the plasmid, they only amplified the gene by PCR and prepared it for cloning, the cloning steps have not been described. Answer page 7: The RUBISCO gene (large chain ribulose-bisphosphate carboxylase gene) is a home gene. It is a positive control of PCR. Content changes are included in the article. Conclusion: The authors claimed that their study demonstrated a great similarity between the THCAS gene of Cannabis Moroccan variety and the reported cDNA precursor gene. However, no sequencing has been conducted to support their claim. Answer conclusion This experience was completed just prior to the COVID pandemic lockdown. So the feasibility of the sequence technique was not feasible."
}
]
}
] | 1
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https://f1000research.com/articles/11-840
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https://f1000research.com/articles/11-1519/v1
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14 Dec 22
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{
"type": "Systematic Review",
"title": "Vaccine approach for human monkeypox over the years and current recommendations to prevent the outbreak: a rapid review",
"authors": [
"Rifat Ara",
"Tajrin Rahman",
"Rima Nath",
"A.M.Khairul Islam",
"Miah MD Akiful Haque",
"Md. Ferdous Rahman",
"Mohammad Hayatun Nabi",
"Mohammad Delwer Hossain Hawlader",
"Tajrin Rahman",
"Rima Nath",
"A.M.Khairul Islam",
"Miah MD Akiful Haque",
"Md. Ferdous Rahman",
"Mohammad Hayatun Nabi",
"Mohammad Delwer Hossain Hawlader"
],
"abstract": "Background: The World Health Organization has declared human monkeypox as a global health emergency on 23 July 2022. This indicates that the outbreak poses a serious risk to global health and requires a united worldwide response to stop the virus from spreading and possibly turning into a pandemic. Vaccines can play a vital role in this context, contributing to pre- and post-exposure prophylaxis. Methods: The aim of our rapid review was to go through the background of the vaccine approach for human monkeypox over the years and to find out what current guidelines are highlighting relating to it. A rapid review with a systematic search and manual searching have been performed here. Results: 22 relevant published articles from MEDLINE bibliographic database and 8 vaccine recommendations from manual searching have been deliberated here. Conclusion: The significant synopsis of this review is that the smallpox vaccine is the only immunization option for monkeypox so far, and it is up to 85% effective to prevent the infection. Third-generation smallpox vaccines are advised over first and second generations due to their minimal side effects. Healthcare providers and lab professionals at risk are on the priority list to get vaccinated, as well as pregnant women or lactating mothers, and immunocompromised or chronically ill patients can get vaccinated if they are surely exposed to the monkeypox infection. Lastly, JYNNEOS/IMVAMUNE is the current most preferable smallpox vaccine that is highly advised for the latest outbreak of human monkeypox but more clinical trials on humans should be conducted to evaluate its safety, efficacy, and adverse events.",
"keywords": [
"Monkeypox",
"Vaccine approach",
"Vaccine recommendations",
"Global health emergency"
],
"content": "Introduction\n\nOrthopoxviruses belong to the Poxviridae, the family of enclosed viruses with large linear, twofold DNA genomes (0.130 kb genome)1. Multiple species of this group can cause serious human illness. Variola virus is the major human pathogen in the family Orthopoxvirus and the causal agent of smallpox. Concerns have been expressed over the use of the variola virus or a similar genetic pathogenic orthopoxvirus as a bioweapon2,3 despite the elimination of smallpox. In addition, there is fear that the variola virus could be discharged inadvertently, such as from unused viral stocks. Variola virus was discovered cold-stored in a United States research laboratory, lending validity to the second possibility4. Orthopoxviruses, such as monkeypox virus, cowpox virus, and strains of vaccinia virus, are emerging zoonoses in many parts of the world5–7. These viruses cause significant diseases in both people and livestock. In reality, both the frequency and severity of human monkeypox virus epidemics have increased6,8,9.\n\nMonkeypox (MPX) is an infectious zoonotic disease largely concealed throughout the smallpox era. It was not recognized as a human disease until the late stages of smallpox eradication campaigns. MPX's concealment might be linked to the comparable clinical manifestations of monkeypox and conventional smallpox, making it difficult to distinguish between the two. How the natural monkeypox virus is sustained in the wild is a significant outstanding question. Currently, natural monkeypox is restricted to humid forest regions in West and Central Africa10. Nevertheless, the disease incidence in the Democratic Republic of Congo has increased since 20016,11. The 2017–18 outbreak of monkeypox in Nigeria spurred the launch of a comprehensive surveillance strategy. Therefore, it is necessary to continue developing and refining orthopoxvirus countermeasures12.\n\nEarly vaccinations used to eliminate smallpox used live, unattenuated vaccinia virus strains derived from calf lymph, like Dryvax13. These vaccinations are no longer manufactured and were replaced by ACAM200014, a cultured cell, live vaccinia virus vaccine, and MVA (Modified Vaccinia Ankara), a replication-deficient (in human tissues) vaccine. Both calf lymph and cell culture vaccinations can cause severe adverse reactions in humans, involving autoinoculation of an eye, widespread vaccinia, eczema vaccinatum, recurrent vaccinia, myocarditis, as well as death13,15,16. In the absence of a more significant global threat posed by orthopoxvirus illness, these safety issues make the broad usage of this vaccine unethical. A substantial number of individuals are inappropriate for ACAM2000 due to security concerns, particularly those with immunological weaknesses and common skin disorders such as dermatitis14. ACAM2000's safety issues encouraged the development of more extensively attenuated third-generation vaccines, such as MVA and Lc16m817. The U.S. Food and Drug Administration has recently approved JYNNEOS, the Barvarian Nordic variant of the MVA vaccine, to prevent smallpox. JYNNEOS protects animals from lethal orthopoxvirus infection, including monkeypox virus disease of nonhuman primates, rabbitpox virus infection of rabbits, and vaccinia virus infection of mice18,19. According to statistics from Africa, the smallpox vaccine is at least 85% effective at preventing monkeypox20. Similar to ACAM2000, MVA lacks known protective targets, and both infections express thousands of genetic variants unlikely to assist in protection21.\n\nFew occurrences of monkeypox outbreaks in humans have been reported outside of the African continent since the 2003 outbreak in 11 states of the USA22. After that a minor uptick was observed in 2017 but the outbreak of 2022 illustrates a distinct situation that may have been predicted because there were early indicators23. 3413 cases with laboratory confirmation and one death have been reported to WHO from 50 countries in five WHO Regions between January 1, 2022 and June 22, 2022. 86% of cases with test confirmation were reported from the WHO European Region24. As of 23rd July 2022, WHO has issued the strongest call to action it is able to in relation to the global monkeypox outbreak by declaring it as a public health emergency of international significance25. By this time, 20,675 confirmed cases of monkeypox and four deaths were reported from 29 EU/EEA countries, as of 25 October 202226.\n\nSeveral vaccine approaches have been applied over the years to prevent the spread of monkeypox infections. A vaccine has recently been approved for monkeypox; however, it is not yet widely available27. Some countries may have smallpox vaccination products on hand that might be used if national guidelines are followed. Depending on the country, any request for vaccination items may be offered in limited numbers through federal authorities. Governments may want to explore immunizing close contacts as a post-exposure prophylactic measure or immunizing select groups of healthcare personnel as a preventative measure. Considering the recent clustered outbreak of monkeypox in several countries and declaring it as a global public health emergency, this rapid review aims to explore different vaccine approaches and plan over the years, challenges and current recommendations to prevent the outbreak.\n\n\nMethods\n\nTo conduct this rapid review, we searched MEDLINE bibliographic database and relevant websites of WHO, CDC, UN, UK Health Agency Security, and GAVI, to identify potential articles describing several vaccine strategies, vaccine efficacy, and vaccine challenges to prevent the monkeypox outbreaks. A simple search term “Monkeypox” AND “Vaccine” was used to find all the pertinent write-ups, and all published documents up to the date of 30 June 2022 were included. Rayyan QCRI tool was used to screen the articles. Primarily we retrieved a total of 291 articles, and duplicate articles (n= 4) were excluded. Two reviewers scrutinized 115 out of 287 articles through independent dual screening of titles and abstracts. Finally, 22 articles were selected after full-text evaluation, and data extraction was considered. The PRISMA flow diagram describes the detailed procedure of article selection (Figure 1). In the case of selecting articles, the availability of the information regarding different vaccine approaches for monkeypox and their outcome were under primary consideration. Vaccine complications and challenges were our secondary objective to include in our review. This review included all relevant published documents, regardless of study type or geographical distribution, such as original articles, perspective papers, review papers, workshop reports, editorial letters, or comments. Because a translator was not available in the team, articles written in languages other than English were excluded.\n\nFor data extraction, key points such as article types, publication year, study design, countries where vaccines have been experimented with, targeted population, name and types of vaccines, their clinical outcome (e.g., efficacy, effectiveness), and challenges for vaccine actualization were assembled and recorded in a structured Excel format. Moreover, we also followed the vaccine recommendations mentioned in the selected articles. We also did manual searching along with the literature review to find out current recommendations by different international health organizations regarding the monkeypox vaccine guidelines. We mainly focused on the advice from the World Health Organization (WHO), Centers for Disease Control and Prevention (CDC), United Nations, UK Health Agency Security, Germany's Standing Committee on Vaccination (STIKO), Gavi, the Vaccine Alliance, and Canada's National Advisory Committee on Immunization (NACI). Recent vaccine recommendations, guidelines, indications, and contraindications were gathered to map out the updated status of monkeypox vaccines. Extracted data was reviewed by a third reviewer to minimize the chances of missing data or biases.\n\n\nResults and discussion\n\nA total of 22 articles have been found that analyzed and described the monkeypox preventive approaches through vaccination (Table 1). Over the years, researchers have explored active and passive surveillance, cohorts, public health investigations, and epidemiological data on monkeypox outbreaks and smallpox vaccination to see the efficacy and effectiveness of different generation vaccines against monkeypox infection. As a result, articles published from 1985 to date were included in our review. Our review perceived that all the articles talked about routine smallpox vaccines of different generations, and no specific vaccine has been manufactured for the monkeypox virus until now.\n\n**Table legends: USA= United States of America, CDC= Centers for Disease Control and Prevention, HCWs= Health care workers, FDA= Food and Drug Administration, DRC= Democratic Republic of the Congo, WTP= Willingness to pay\n\nThere are five articles published in 1980s that mainly investigated individuals of the African region who were routinely vaccinated against smallpox during their childhood and later exposed to the monkeypox virus. Among these five articles, Arita I. et al. assessed the special surveillance of tropical rain forests of West and Central Africa between 1970 to 1981 and 1982 to 1983, where a 13% case-fatality rate was achieved, and all of them happened to unvaccinated youngsters. Moreover, the secondary attack rate in unvaccinated contacts was around 15%28. The other four articles analyzed data on the monkeypox outbreak in Zaire of the Democratic Republic of Congo (DRC) over five years (1980–1984). They concluded that the standard routine smallpox vaccine was 85% effective in preventing monkeypox29, and the secondary attack rates were 0.110 in unvaccinated contacts living in the same households29,30.\n\nAfter the 1980s outbreak of monkeypox in African territories, a long break took place studying preventive measures for monkeypox until the outbreak emerged in the USA in 2003. The first indication of community-acquired monkeypox in the United States was reported by the Centers for Disease Control and Prevention (CDC) at the beginning of June 2003, and CDC advised the vaccinia smallpox vaccine be used as it was established to be 85% effective against monkeypox32. Seven articles have discussed the effectiveness of past smallpox vaccination among US citizens to prevent monkeypox, where almost all the studies found positive outcomes of vaccination except one. A retrospective analysis of clinical reports and active and passive surveillance of suspected monkeypox cases was done in 2005, where bivariate and multivariate analysis found no association of disease severity or hospitalization with previous smallpox vaccination33. Vaccinated subjects showed good protection and less pronounced clinical signs or symptoms against the onset of monkeypox-induced disease in the rest of the studies33–35. Another significant public health investigation was done in 2005, where 94% of the monkeypox-exposed healthcare workers tested positive for anti-orthopoxvirus IgG antibodies as they had a previous history of smallpox vaccination36. Jamieson D.J. et al. and Cono J. et al. are the two articles that put forward the opinion regarding smallpox vaccination during pregnancy31,36. According to them, the smallpox vaccine is considered category C (the human fetal risk of the drug as unknown due to no human studies or positive results in animal studies) approved by the US Food and Drug Administration (FDA), and minor risk to the fetus from vaccinia smallpox immunization during pregnancy exists that may result in premature birth, fetal and neonatal death. Nevertheless, given the potentially fatal risk of monkeypox infection, exposed women were urged to get the smallpox vaccine regardless of whether they were pregnant.\n\nAnother significant finding of our review was that despite having multiple animal trials of the vaccine directly against monkeypox, there is no sufficient evidence of clinical trials on humans yet. In recent times, a phase I/II randomized, double-blind, comparative clinical trial of LC16m8 (an attenuated cell culture–adapted Lister vaccinia smallpox vaccine) has been conducted on humans to compare the safety and immunogenicity of LC16m8 with the Dryvax vaccine38. This trial showed that LC16m8 is a feasible next-generation vaccination alternative to first-generation smallpox vaccines to prevent human monkeypox, at least in high-risk groups. Although, its clinical efficacy against human monkeypox has not yet been determined. A clinical trial of a new generation IMVAMUNE® smallpox vaccine is ongoing to evaluate its safety and efficacy in preventing monkeypox among healthcare workers (HCWs) in the Democratic Republic of the Congo39.\n\nRight now, the Strategic National Stockpile (SNS) has three smallpox vaccines, among which ACAM2000® and JYNNEOSTM (also known as IMVAMUNE or IMVANEX) are the only two licensed smallpox vaccines in the United States40. It has been found that the only smallpox vaccination effective in the people of the DRC was the live vaccine inoculation (Dryvax and ACAM2000)41. Monkeypox risk was 5.21 times lower in vaccinated individuals compared to unvaccinated individuals, showing that more than 80% protection was maintained for over 30 years41. A prospective cohort study was performed in 2019 to follow up the health workers of the DRC who have received two doses of the third-generation smallpox vaccine (IMVAMUNE) due to reporting so many adverse events of first- and second-generation smallpox vaccine42. Third-generation smallpox vaccine was better than the first- and second-generation. Though a clinical trial is ongoing to evaluate the safety and efficacy of the IMVAMUNE vaccine against human monkeypox among healthcare workers, an online based cross-sectional study was conducted among Indonesian health workers to evaluate the acceptance and willingness to pay (WTP) for the vaccine, where 96% of the participants expressed the acceptance of free vaccination43. A case study in Singapore revealed that 64% of close contacts recovered rapidly from signs and symptoms of monkeypox due to accepting the ACAM2000 vaccine (Sanofi Pasteur Biologics Co) immediately after contact tracing44.\n\nThe rate is inferior if we look into the recent smallpox vaccination status. In order to prevent smallpox from reemerging, WHO kept a stockpile of 200 million doses in 1980. However, when smallpox did not resurface in the late 1980s, 99% of the stockpile was destroyed45. By 2019, the United States had received 269 million doses of ACAM2000 and 28 million doses of MVA45,46, but by the time the 2022 monkeypox outbreak began, only 100 million doses of ACAM2000 and 65,000 doses of MVA remained in the stockpile48. One of our included articles showed that only 10.1% of Nigeria's population had received the smallpox vaccine as of 2016, and the serologic immunity level was 25.7% among those who had received the vaccination compared to 2.6% in the general population49. However, worldwide vaccination data is not known yet. Finally, there is one article that criticized the cost-benefit of mass vaccination. Bankuru S.V. et al. describe a compartmental epidemiological model and game theory approach that evaluated vaccination decision-making of a community50. The model quantifies the smallpox vaccine's costs and advantages. This study determined that the ideal vaccination rate is approximately 0.04, meaning people should get vaccinated once every 25 years. Additionally, they discovered that monkeypox is preventable and can be eliminated through vaccination in a semi-endemic equilibrium (an infection that spreads only part of the year in a specific area). However, vaccination alone cannot wholly eradicate monkeypox in an equilibrium where it is entirely endemic50.\n\nAfter manual searching for vaccine recommendations, several guidelines were put in place suggested by different international health organizations (Table 2). According to WHO, mass vaccination for monkeypox is not required so far. They also advise administering a suitable second or third-generation smallpox vaccine to contacts as post-exposure prophylaxis (ideally within four days of initial exposure) and healthcare workers at risk as pre-exposure prophylaxis51. CDC recommends JYNNEOS™ for certain laboratory workers and clinic teams who are susceptible to virus exposure. JYNNEOS™ is usually issued in two doses, given four weeks apart. People who have received other types of smallpox vaccine in the past might be considered for one dose only. Booster doses are recommended every 2 or 10 years if a person remains at continued risk for exposure to orthopoxviruses. JYNNEOS™ is recommended for individuals exposed to the monkeypox virus regardless of concurrent illnesses, pregnancy, breastfeeding, or poor immune system52. On the other hand, the CDC also advised ACAM2000 immunization for military personnel and lab workers only, but it is not suggested for any immunocompromised health condition (such as diabetes or pregnancy) as ACAM2000 has the potential for more side effects and adverse events than JYNNEOS52,53. The Vaccine Alliance-GAVI has recommended the vaccinia smallpox vaccine over the first generation and suggested increasing the availability worldwide due to the monkeypox outbreak55. Other national health organizations, such as the National Advisory Committee on Immunization (NACI)-Canada, the U.S. Department of Health and Human Services (HHS), and Germany's Standing Committee on Vaccination (STIKO), have also suggested the latest smallpox vaccine (JYNNEOS/IMVAMUNE/IMVANEX) to fight against the recent outbreak of human monkeypox55–57.\n\n\nConclusion\n\nAs the rapidly spreading monkeypox outbreak in 2022 represents a global health emergency, the WHO labeled it as a \"public health emergency of international concern (PHEIC)\" and asked for an international response to collaborate on sharing vaccines and treatments59. So, governments worldwide should come forward immediately to impose preventive measures, including screening, isolation, and vaccine prophylaxis where necessary.\n\nThe limitation of our review was that we only searched the MEDLINE database due to the target of rapid review within a short period of time. A systematic review can also be performed in this regard, especially on vaccine clinical trials. Despite having some deficiencies, this rapid review on vaccine approach and recommendations concluded with several significant points, including:\n\nThe smallpox vaccine has been the only immunization option for human monkeypox till now. There is no specific vaccine manufactured only for monkeypox yet.\n\nThe smallpox vaccine is up to 85% effective in preventing monkeypox infection and it can provide protection for a very long period.\n\nThere are three categories of smallpox vaccine. The first-generation vaccine is not available in the market anymore. The third-generation vaccine is recommended over the first and second generation due to fewer side effects and adverse events.\n\nSmallpox vaccine is recommended for all monkeypox virus exposed individuals regardless of pregnancy, chronic illnesses, or poor immunological conditions.\n\nJYNNEOS/IMVAMUNE is the latest third-generation smallpox vaccine that almost all international health organizations have mostly recommended. A human clinical trial of this vaccine is currently ongoing, and the results could offer information which would be very much helpful to evaluate the safety and efficacy of the vaccine. More trial studies should be conducted in the future to find out the accuracy.\n\nThe vaccine is highly recommended as pre-exposure prophylaxis to all healthcare workers at risk and contact as post-exposure prophylaxis, but mass vaccination is not required until now.\n\nAdditionally, human monkeypox is preventable and can be eradicated through vaccination alone in a semi-endemic equilibrium but not in a fully endemic equilibrium.\n\nThe approval for this rapid review has been granted by the Ethics Review Committee of North South University, Bangladesh on 2nd July 2022. Reference number: 2022/OR-NSU/IRB/1001. This study did not directly involve any human participant; therefore, consent was not required.",
"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: PRISMA checklist and flowchart for ‘Vaccine approach for human monkeypox over the years and current recommendations to prevent the outbreak: a rapid review’. https://doi.org/10.6084/m9.figshare.21423666.v164\n\n\nAcknowledgements\n\nAn earlier version of this review has been published in medRxiv: http://dx.doi.org/10.1101/2022.09.29.22280481.\n\n\nReferences\n\nMoss B: Smallpox vaccines: targets of protective immunity. Immunol Rev. 2011; 239(1): 8–26. PubMed Abstract | Publisher Full Text | Free Full Text\n\nSmith GL, McFadden G: Smallpox: anything to declare? Nat Rev Immunol. 2002; 2(7): 521–7. PubMed Abstract | Publisher Full Text\n\nNoyce RS, Lederman S, Evans DH: Construction of an infectious horsepox virus vaccine from chemically synthesized DNA fragments. PLoS One. 2018; 13(1): e0188453. 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PubMed Abstract | Publisher Full Text\n\nHammarlund E, Lewis MW, Carter SV, et al.: Multiple diagnostic techniques identify previously vaccinated individuals with protective immunity against monkeypox. Nat Med. 2005; 11(9): 1005–11. PubMed Abstract | Publisher Full Text\n\nKarem KL, Reynolds M, Hughes C, et al.: Monkeypox-induced immunity and failure of childhood smallpox vaccination to provide complete protection. Clin Vaccine Immunol. 2007; 14(10): 1318–27. PubMed Abstract | Publisher Full Text | Free Full Text\n\nFleischauer AT, Kile JC, Davidson M, et al.: Evaluation of human-to-human transmission of monkeypox from infected patients to health care workers. Clin Infect Dis. 2005; 40(5): 689–94. PubMed Abstract | Publisher Full Text\n\nCono J, Cragan JD, Jamieson DJ, et al.: Prophylaxis and treatment of pregnant women for emerging infections and bioterrorism emergencies. Emerg Infect Dis. 2006; 12(11): 1631–7. PubMed Abstract | Publisher Full Text | Free Full Text\n\nKennedy JS, Gurwith M, Dekker CL, et al.: Safety and immunogenicity of LC16m8, an attenuated smallpox vaccine in vaccinia-naive adults. J Infect Dis. 2011; 204(9): 1395–402. PubMed Abstract | Publisher Full Text | Free Full Text\n\nIMVAMUNE® Smallpox Vaccine in Adult Healthcare Personnel at Risk for Monkeypox in the Democratic Republic of the Congo. Reference Source\n\nCenters for Disease Control and Prevention (CDC): Vaccines. The Strategic National Stockpile (SNS,vaccines in the United States, 2019. Reference Source\n\nRimoin AW, Graham BS: Whither monkeypox vaccination. Vaccine. 2011; 29 Suppl 4(Suppl 4): D60–4. PubMed Abstract | Publisher Full Text | Free Full Text\n\nPetersen BW, Kabamba J, McCollum AM, et al.: Vaccinating against monkeypox in the Democratic Republic of the Congo. Antiviral Res. 2019; 162: 171–7. 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): 6800–6. PubMed Abstract | Publisher Full Text\n\nYong SEF, Ng OT, Ho ZJM, et al.: Imported Monkeypox, Singapore. Emerg Infect Dis. 2020; 26(8): 1826–30. PubMed Abstract | Publisher Full Text | Free Full Text\n\nThe World Health Organization: Operational framework for deployment of the World Health Organization smallpox vaccine emergency stockpile in response to a smallpox event. Reference Source\n\nEmergent BioSolutions Awarded 10-Year HHS Contract to Deliver ACAM2000®, (Smallpox (Vaccinia) Vaccine, Live) Into the Strategic. 2019. Reference Source\n\nBAVARIAN NORDIC ANNOUNCES U.S. FDA APPROVAL OF JYNNEOSTM (SMALLPOX AND MONKEYPOX VACCINE, LIVE, NON-REPLICATING) FOR PREVENTION OF SMALLPOX AND MONKEYPOX DISEASE IN ADULTS. 2019. Reference Source\n\nHHS Orders 2.5 Million More Doses of JYNNEOS Vaccine For Monkeypox Preparedness. 2022. Reference Source\n\nNguyen PY, Ajisegiri WS, Costantino V, et al.: Reemergence of Human Monkeypox and Declining Population Immunity in the Context of Urbanization, Nigeria, 2017-2020. Emerg Infect Dis. 2021; 27(4): 1007–14. PubMed Abstract | Publisher Full Text | Free Full Text\n\nBankuru SV, Kossol S, Hou W, et al.: A game-theoretic model of Monkeypox to assess vaccination strategies. PeerJ. 2020; 8: e9272. PubMed Abstract | Publisher Full Text | Free Full Text\n\nThe World Health Organization: Vaccines and immunization for monkeypox: Interim guidance, 2022. Reference Source\n\nCenters for Disease Control and Prevention (CDC): Smallpox/Monkeypox VIS. 2022. Reference Source\n\nCenters for Disease Control and Prevention (CDC): Monkeypox and Smallpox Vaccine Guidance. 2022. Reference Source\n\nCenters for Disease Control and Prevention (CDC): Considerations for Monkeypox Vaccination. 2022. Reference Source\n\nGAVI: Five things you need to know about monkeypox. 2022. Reference Source\n\nTeresa Wright: Monkeypox: Vaccine recommended for Canadians at high risk of exposure. 2022. Reference Source\n\nThe U.S. Department of Health and Human Services (HHS): HHS Announces Enhanced Strategy to Vaccinate and Protect At-Risk Individuals from the Current Monkeypox Outbreak. 2022. Reference Source\n\nDW: Monkeypox: German panel recommends vaccine for risk groups. 2022. Reference Source\n\nReuters: WHO declares global health emergency over monkeypox outbreak. 2022. Reference Source\n\nJezek Z, Grab B, Szczeniowski MV, et al.: Human monkeypox: secondary attack rates. Bull World Health Organ. 1988; 66(4): 465–70. PubMed Abstract | Free Full Text\n\nNalca A, Rimoin AW, Bavari S, et al.: Reemergence of monkeypox: prevalence, diagnostics, and countermeasures. Clin Infect Dis. 2005; 41(12): 1765–71. PubMed Abstract | Publisher Full Text\n\nKalthan E, Tenguere J, Ndjapou SG, et al.: Investigation of an outbreak of monkeypox in an area occupied by armed groups, Central African Republic. Med Mal Infect. 2018; 48(4): 263–8. PubMed Abstract | Publisher Full Text | Free Full Text\n\nWhitehouse ER, Bonwitt J, Hughes CM, et al.: Clinical and Epidemiological Findings from Enhanced Monkeypox Surveillance in Tshuapa Province, Democratic Republic of the Congo During 2011-2015. J Infect Dis. 2021; 223(11): 1870–8. PubMed Abstract | Publisher Full Text\n\nAra R, Haque MMA, Nabi MH, et al.: Vaccine approach for human monkeypox over the years and current recommendations to prevent the outbreak: a rapid review. figshare. 2022. http://www.doi.org/10.6084/m9.figshare.21423666.v1"
}
|
[
{
"id": "173503",
"date": "24 May 2023",
"name": "Chandrakant Lahariya",
"expertise": [
"Reviewer Expertise Vaccines",
"Primary health care",
"Outbreaks",
"epidemics and pandemics",
"Diabetes",
"Hypertension",
"Health systems",
"Health Policy"
],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nIntroduction can mention more about monkeypox virus and disease, the first paragraph can be shortened or omitted. Explain in the context of the current outbreak. Proceed with respect to virus and clinical scenario in brief, followed by past and current outbreak scenario followed by vaccinations and vaccines in brief and objective in up to 2 sentences.\n\nIn objective of the study, the research question is unclear and very limited information was given on the approved vaccines.\n\nWhy was the geographical distribution not considered? This has a huge bearing with respect to vaccination and vaccine logistics and can hugely impact disease outcome.\n\nTables can be made more concise if possible, especially Table 2.\n\nThe article mentions the need for an international response, but it doesn't go into detail about why collaboration is essential or what particular advantages might be realized through it. The case for quick action would be strengthened by outlining the justification for the necessity of a global response and the possible benefits of international cooperation.\n\nThe article makes no mention of when governments should step forward and implement preventive actions. It would be easier for people to comprehend what needs to be done right away if there was a sense of urgency or a time frame for the response.\n\nThe author may consider to read and use the following recent papers in discussion section: Rajkhowa et al., (2023)1, Lahariya et al., (2022)2.\n\nThe challenges should be mentioned as a separate heading for clear understanding.\n\nPlease mention actionable recommendations and findings of the review on the conclusion, in sentence form instead of bullet form.\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? Not applicable\n\nAre the conclusions drawn adequately supported by the results presented in the review? Partly",
"responses": []
},
{
"id": "179843",
"date": "11 Jul 2023",
"name": "Zhenhao Fang",
"expertise": [
"Reviewer Expertise mRNA vaccine",
"Mpox research",
"Infectious Disease",
"Vaccine characterization"
],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nAra et al. reviewed dozens of MEDLINE articles studying smallpox/monkeypox vaccines and 8 vaccine recommendations for monkeypox. The review provides a quick overview of vaccine guidelines and progress made in clinical studies characterizing monkeypox vaccine. Suggestions for this review article is summarized below.\nAuthors may want to mention the WHO recommendation made in November 2022 to adopt “Mpox” as the term used to refer to monkeypox disease.\n\nAdditional clinical trials and preclinical studies that support the authorization of JYNNEOS and ACAM2000 should be cited, such as Phase 3 trial by Pittman et al.,1 and Monkeypox virus challenge in Macaques by Hatch et al.2\n\nThe take-home messages of each article in table 1 and 2 could be refined and simplified.\n\nSeveral research groups have made progress in developing Mpox-targeting mRNA vaccines. Examples of these studies include: 1) a brief report by Fang et al.3 and 2) a preprint by Freyn et al.4. A brief discussion of this recent progress is highly recommended.\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": []
}
] | 1
|
https://f1000research.com/articles/11-1519
|
https://f1000research.com/articles/11-1515/v1
|
13 Dec 22
|
{
"type": "Case Study",
"title": "Beyond tradition: using systemic functional linguistics in TED talks",
"authors": [
"Hai Tang",
"Lu Bai",
"Shuanghui Chen",
"Lu Bai",
"Shuanghui Chen"
],
"abstract": "TED (Technology, Entertainment, Design) is an internationally based open access platform where professionals from various fields are invited to give speeches, and to share ideas with like-minded people. In recent years, topics related to the coronavirus disease (COVID-19) or the COVID-19 pandemic have become the most popular speech topics on TED. This paper selects speech-related topics on TED during the pandemic, by using systemic functional linguistics to study how discourses are expressed on the platform. Unlike traditional textual analyses, content on TED are normally verbalized, requiring original texts to be presented in an understandable format. As a result, this paper focuses on the functional innovation of new forms of expression on TED. In other words, the functional perspective of the presentation in relation to COVID-19 based topics may rely on the explanation of the meta-meaning of the keywords, posting images and other techniques as auxiliary means for expression, to some extent. These auxiliary techniques offer traditional systemic functional linguistics new meanings in terms of ideational and interpersonal aspects.",
"keywords": [
"Systemic Functional Linguistics",
"TED",
"COVID pandemic",
"Ideational meaning",
"Interpersonal meaning"
],
"content": "1. Introduction\n\nSpeech has multiple forms, and is a kind of communication between presenters and audiences. With the continuous improvement of new media technology, text-based speeches are presented by speakers through multimodal pragmatic means, giving listeners new ideas, and greatly helping listeners to understand the speech topics at the same time. The need for novel topics, along with in-depth explanations, has led to “pragmatic functional innovation” (Gu, 2004:82) in the process of the presentation.\n\nStudies on speeches have found that sticking to text only cannot satisfy the demand for the deployment of systemic functional linguistics. The use of modern language in words and sentences is stylishly casual and irregular, for instance, the appearance of net-language has constantly refreshed people’s understanding of the original meaning of the word (Li, 2019), while a series of scientific vocabularies (e.g., ‘drive’ as a computing word, ‘monitor’ as a medical word) have to be understood through proper explanations within particular contexts (Gao, 2012). The latter, like topics discussed during the coronavirus disease (COVID-19) pandemic on the TED (Technology, Entertainment, Design) platform, is a case in point. In this sense, the introduction of pictures, PowerPoint, audio and videos may potentially facilitate the effective interactivity in the speech (Feng, 2011).\n\nThis paper utilises topics during the COVID-19 pandemic on TED to reinterpret systemic functional linguistics. It argues that topics such as COVID-19 on TED incorporate an updated utilization of systemic functional linguistics. Such an updated utilization, to some extent, lies in excavating the usage of the term “functional”. In other words, words or phrases in systematic functional linguistics contain two-layer meanings: the meaning in lexical grammar and the meaning in semantics (Zhao, 2016:642); within different contexts, the latter can be manifested as variability, deriving ideational meaning or refracting interpersonal meaning in different cases.\n\nIn order to explain how the ‘functional’ reinterprets the theory, this paper raises the following questions: how did presenters deal with subjectivity in TED speeches and to what extent can the lexis be treated in a more functional way than they were used in text?\n\nTo answer these questions, this paper is structured into five sections. Besides the above contents set as an introductory section, the next section explores the historical development of the theory to show how systemic functional linguistics has been broadly used for various case studies. After a brief introduction of TED in the third section, the fourth section selects topics which were presented on TED during the COVID-19 pandemic, with the aim of analyzing the pragmatic functions of semantics and semiotics of the speeches so as to explore how these topics bring ideational meaning or interpersonal meaning, and thus, to sort out any difference between the old theory and its new usage. Section five concludes that the term ‘functional’ is embodied in both the fractal of the meaning (Zhao, 2016) and relevance of the context (Cai, 1997), providing new pragmatic values for systemic functional linguistics.\n\n\n2. The broad use of systemic functional linguistics in practice\n\nSystemic functional linguistics were established by Michael Alexander Kirkwood Halliday in the late 1950s (Chang, 2012; Gong, 2011; Yang, 2015). Halliday (1970) believed that textual analyses of each part in a setting topic (literal or audiovisual) correspond to a meta-function, that is, textual, ideational and interpersonal functions are interrelated in sentences or passages to confirm the focus point of the theory. One of Halliday’s representative works, Studies in English Language, was published to show the meanings in semantics and lexical-grammar, and the relation in between the two meanings was explained as realization instead of constituency, identifying that meta-function and strata were the most prominent features of the language (Yang, 2015:24).\n\nThere are many researchers who have been expanding the usage of the two features based on different research topics. For example, in China, stratificational grammar has been particularly applied to educational and communicative fields. In Genre Studies in Systemic Functional Linguistics, Gong Changhua (2011) researched discourse genres and found that the study of the structure of linguistics and the characteristics of language expression explores the reasons for using the formation of the language. For instance, since speaking, reading, listening, and writing are the four fundamental skills for language learners, practicing listening and reading skills while immersed in a situation that is relevant to their learning goals might aid students in understanding the target language more rapidly (Cai, 1997:18). Likewise, the speaking and writing courses aim at cultivating a shared cognitive environment so that students are purposely trained to express both sentence meaning and utterance meaning clearly (Gu, 2004:82).\n\nLou Qi and Zhang Wei’s (2009) research focused on the interpersonal function of meta-pragmatics. They believed that an author could clearly speak out thoughts or ideas via meta-discourse so as to build up interpersonal relations with his readers. What Lou and Zhang highlighted is the tone, the clause, or the attitude the author chose to communicate with his readers, not merely the content he wrote, as the former could constitute an independent linguistic structure. Thus, Lou and Zhang proposed a ‘dual-order linguistic perspective’ in their work that helped describe the conversational implicatures and the communicative intention of the author.\n\nIt can be seen from the above case studies that language can be read in three levels: linguistic material (in written texts or audiovisual formation), lexical grammar and context. While scholars are likely to emphasize the central position of the lexical grammar when they research on teaching and communication cases, as Xu Laijuan argues:\n\n“Studies of the Linguistics shall be notified that the focus point lies in understanding lexical grammar of the passage. It is through explanation of the lexicon, or the grammatic usage of the words, phrases or sentences can students learn meanings of the passage, using the language to communicate with each other and make their expressions understandable.” (Xu, 2014:104)\n\nThe above studies additionally illustrate a coherent relationship between semantics and the context. In other words, one can convey the meaning through certain contexts. However, with the continuous development of using formations for expression, there might not necessarily a corresponding coherence between lexicon meaning and pragmatic meaning. In The Limitations of Systemic Functional Linguistics: The Case of Online Expressions, Li Xingying discussed the lexicon and sound features of online languages and found that the meaning of online words or phrases could hardly be explained in lexicon meaning, rather, they represent discursive expressions of the netizens (Li, 2019:145).\n\n“The net words contain two realizations of the semantic meaning. Unlike normal stratifications set by three levels, netizens form their discourses through the sound, which can be translated into different characters. In other words, the meaning of the online expressions is firstly reflected in the sound layer, and then it is understood in lexical grammatic system, shaped as homonyms or abbreviations.” (Li, 2019:147)\n\nThe ‘irregular’ understanding of the meaning coined by netizens suggest that analyses of the context is rather important; while in the process of understanding the meaning, the notion of the ‘fractal’ (Zhao, 2016) in shaping the meaning should also be noted, as the meaning (ideational, interpersonal, and textual) may have the potential to be expanded or projected.\n\nThe particularity of the above online language reflects how people use language on social media to build or change interpersonal relations (Locher & Graham, 2010:2). Such particularity can be seen as a new understanding of the interpersonal meaning in communication. The new understanding indirectly elucidates the motivations and mechanisms underlying the use of the meta-function in systemic functional linguistics, as Kristin Davidse and Anne-Marie Simon-Vandenbergen cited:\n\n“The notion of ‘interpersonal’ meaning, which this thematic issue is concerned with, has to be situated within the ‘metafunctional’ theory which Halliday and his students have been developing since the mid 1960’s. The basic tenet is that all main linguistic systems (such as transitivity, modality and thematic organization, to name but a few) can be analyzed as serving one of three kinds of very general functions, or ‘META’-FUNCTIONS: the ideational, the interpersonal and the textual.” (Kristin & Anne-Marie, 2015:3)\n\nThis concept of interpersonal meaning had a big influence on Hallidayan, especially on how they used the notion to analyse discourse and language change. For example, He Fangzhi (2009) argued in her Meta-functional Analyses of the Humor in Verbal Communication, that vocabulary could be used for expression in general, while she took Friends as an example to note that the use of the verbs could be somehow exaggerated to produce humorous effects in expression.\n\nLikewise, Hakibou Abdoulaye’s study on Half of a Yellow Sun (2022) aimed to describe the interpersonal meaning of the narrative through revealing interpersonal realizations by looking at the mood system and modality. Hakibou thus argued that declarative mood could be used as an expression of certainty so as to show the function of textual semiotics.\n\nAs can be seen from the above examples, either humorous expressions or declarative moods, they are the functional usage of the systematic functional linguistics, as Chang Chenguang argues:\n\n“Expression can be seen as a way of ‘doing things’, which has semantic potential. Since systematic functional linguistics emphasizes function rather than structure of the language, to some extent, the expression can also be embodied with ‘behavioral potential’.” (Chang, 2012:19)\n\nIn recent years, expressions embodied with behavioral potential have become a normal trend in international business meetings. Liu Ping, Chen Jialiang and Yang Linlin’s research focuses on Interpersonal Functions of Metapragmatic Expressions in International Business Meetings (2022), and they suggest that such meetings are goal-oriented activities; the process of the communication is constrained by multiple factors such as business organization, multiculturalism and language proficiency of the communicators. While the researchers have found that linguistic resources and pragmatic strategies are actively used to manage and regulate business communication, all of which can be seen as behavioral-based functional potential.\n\nFrom the above illustrative description of the meta-function and semantic exploration presented by scholars, there are two concepts that need to be noted. The first is that, the lexical meaning can be expanded in certain context, due to the fractals of the meaning in systemic functional linguistics. In addition, interpersonal meaning emphasizes auxiliary functions such as how speakers perform “speech act” with certain attitude, mood and purpose to reflect the real meaning. The two concepts: “fractal” and “auxiliary functions” used in systemic functional linguistics will be in-depth explored by the analyses of the COVID-19-related topics on TED in the following section, and the aim is to break down the traditional understanding of systematic functional linguistics in the grammatical and empirical categories.\n\n\n3. An introduction to TED\n\nTED is a nonprofit organization devoted to spreading ideas, usually in the form of short talks. It was established by Richard Saul Wurman who was a popular graphic designer (Hong & Liang, 2013:12). TED was founded in 1984 as a result of the TED Conference. The conference used to draw a lot of intellectually interested individuals who wanted to express their thoughts in a public setting. In 2005, TED introduced the TED Global Conference and promoted topics on a wide range of new themes, extending to embrace the fields of education, culture, business, and the arts in addition to technology, entertainment, and design. Since 2006, TED has created a collection of speeches that have been captured over the years called TED Talks. These talks are posted to the internet and made freely downloadable and viewable to audiences across the world. As of March 2013, there were more than 1400 TED Talks videos available on the organization’s official website, which helped TED progressively gain popularity (Hong & Liang, 2013:12-13).\n\nFirstly, topics in TED involve educational meaning, however, they are quite different from traditional lessons given in classrooms, nor are they as similar as online learning portals, take NetEase as an example of a cloud classroom, it focuses on teaching the knowledge, but the teaching target is in selling the courses. Secondly, topics in relation to technology, culture or economics involve in-depth knowledge, they enlighten people to share ideas in terms of criticism and creation. For instance, Ken Robinson’s presentation, Do Schools Kill Creativity? was lauded by online users, and it sparked a discussion over the various definitions of the term “creativity.” In other words, by looking at the role of the school from various perspectives, netizens questioned the wisdom of its “creativity” policy, claiming that the idea of “creativity” would be acceptable in educational settings and should be promoted across many areas.\n\nToday, TED shares ideas from a very broad spectrum, from global issues to business and science in beyond 100 languages, just as the slogan goes on its official site, “Ideas Worth Spread”. The following section will take topics during the COVID-19 pandemic as representative of novel speeches, and to see how such speeches are presented on TED, how speakers expressed their viewpoints, and how the feature of their speeches and presentations relate to the use of systemic functional linguistics.\n\n\n4. The use of functional approach in the COVID-19 based topics on TED\n\nThe features of TED introduced above see it as a communicative platform in which speakers discuss topics to exchange ideas, express viewpoints on the world and build interpersonal interactions with audiences. In order to achieve a communicative purpose, speakers are likely to adopt various presentation techniques and methods to enable audiences to understand their viewpoints and resonate with them. In this regard, the mood and modality systems which are used as functional approaches seem to be outdated to some extent, that is to say, the means of the realization of the meta-discourse can be diverse in speech-based topics.\n\nIn English, semantics is used to express meanings under particular contexts. The representative traditional Semiotic Triangle Theory holds that meaning associates word with referent in the minds of the speakers through conventional concepts so as to build interactive relations, or, to set reasonable ways for the hearers to understand the meaning of the word (Gu, 2004:80). Take TED, in Dina Katabi’s A New Way to Monitor Vital Signs (that Can See Through Walls), the presenter said: “And we are going to monitor him as he moves.” The term ‘monitor’ here, as a verb, is used in medical context, the meaning of it is to use medical device to trace the patient’s health status.\n\nThe above example is not simply about the definition of the term in semantics; but it argues that since the lexical system has been always in constant flux over the time, then determining the exact meaning of a word needs to put the word into its semantic fields (Cai, 1997:15).\n\nThe COVID-19 pandemic has been caused by a global disease that has been described by several international organizations or media as the most severe crisis since World War II1 (Guan, 2020), and it can be seen as the worst public health event in history (Wang & Lei, 2022:30).\n\nSeeing the opportunity, TED has also grasped the topic during the pandemic and invited various experts to come to the platform to give speeches, providing detailed explanations on the cause, the treatment and the prevention of the epidemic.\n\nDavid Heymann is a professor of infectious disease epidemiology at the London School of Hygiene and Tropical Medicine. He gave a speech What We Do (and Don't) Know About the Coronavirus on TED on February 27, 2020.\n\nFrom reading the title we know that David aimed at explaining the term “coronavirus” and, in his lecture, David frequently mentioned the words “disease”, “infection”, “virus”, “people” and so on, trying to demonstrate what caused the coronavirus and how these words relate to COVID-19 during the pandemic.\n\nFor instance, in explaining the term “disease”, David used the following methods. The first is to introduce the symptom: “This looks like a very mild disease, like a common cold, in the majority of people.” The second is to define the symptom: “pulmonary disease”. The description of the two symptoms distinguishes coronavirus from the common cold. Meanwhile, David proposed warning messages in his talk – “It’s clear we know how it transmits, we don’t know how easily it transmits in humans, in communities or in unenclosed areas” so as to imply that coronavirus related to the term “contagion”.\n\nIt can be seen that, when David tried to define the term “disease”, he focused on how to connect the word with coronavirus. In other words, the meaning of ‘disease’ can be defined variously due to its diverse symptoms and the hints of the harmfulness in transmission; all of which can be seen as contextual factors to impact on understanding of “coronavirus”.\n\nAs a result, expression of the lexical information in a speech is different from the one explained in the dictionary. The difference may come from the context that the speaker sets for the topic. Thus, the function of the context is to help listeners understand specific meanings of the common words.\n\nIn How COVID-19 Transformed the Future of Medicine, Daniel Kraft mentioned the adjective “medical” in different paragraphs. At the beginning of the speech, Daniel said, “… the convergence of ever smaller interconnected devices now riding 5G is creating not just an Internet of Things but an Internet of Medical Things”, connecting pharmaceutical reform with digital health. In addition, the sentence that “Now exponential technologies packed into our smart devices are becoming increasingly medicalized” again associated medical industry with smart devices. Daniel even described the diagnosis of COVID-19 patients as a “medical selfie”, further enhancing the intelligent function of the medical industry.\n\nFrom reading the above sentences we can see that, in the process of explaining the word “medical”, Daniel added a lot of new information such as “Internet of Medical Things”, “medicalized”, and “medical selfie”. These new messages are not just acted as rhetorical elements, but they delineated the contextual scope of the topic, and encapsulated Daniel’s viewpoint that the digital health system achieved “the real potential to reshape and scale health care at our pandemic age”.\n\nAs can be seen from the above analyses of the two words (“disease”, and “medical”), the determining of semantics should consider their meaning under certain context. That is to say, the meaning of the two words in their vocabulary system may be different from the ones defined in different contexts, while listeners can understand their actual meaning because they too, place the words into the “semantic fields” (Gu, 2004:81) in conjunction with the context, especially verbal context to get the speakers’ meaning.\n\nAs TED is a speech-based platform, speakers use words and sentences to present their ideas, which also reflect their conversational implicatures with communicative values. In this sense, determining semantic meaning should also consider the role of nonverbal contextual factors, for instance, the speakers’ speech act.\n\nMatt Walker gave a speech 4 Ways the COVID-19 Pandemic Changed the Way We Sleep, which only lasted two minutes. While Matt posted several pictures during his speech, he clearly explained four changed ways of sleep in COVID-19 pandemic.\n\nAt the very beginning, Matt mentioned that “the COVID pandemic changed sleep in at least four different ways: “quantity, quality, timing and dreaming”. By posting a photo, Matt effectively communicated to the audience the erratic nature of sleep. In Matt’s picture, the clock and pillow were located at the upper left and right corners respectively. An image like that illustrates how individuals typically go to bed at the appropriate time. However, the sun and moon were in the bottom left corner of the image in the same frame, hinting at the phenomenon of people sleeping irregular hours. The fabric that corresponded to the pillow was scattered, with colours in dark green, bright purple, dark blue, and bright yellow interspersed to create contrast and further hint at the scenario of people sleeping restlessly. Likewise, Matt used another two pictures to indicate the change in sleep quality during the COVID-19 pandemic: one represented the normal state of a woman who fell asleep at night. Dark blue served as the image’s basis colour, dark green was employed for the pillow as comforter, and the woman was dozing on her side. Yet Matt’s next picture shifted the color of the bedroom mostly from dark blue to light gray, along with a change of her sleeping position, all of which indicated a worse trend in sleep quality. Then Matt commented: “indeed, in the US, almost 60 percent of people felt that the quality of their sleep had become worse during the pandemic.” Thus, Matt’s emphasis on “60 percent of people” can be seen as presupposition to claim his explanation of the word “change” which was given the meaning of getting worse.\n\nUnlike the speeches illustrated in the previous section, here Matt used pictures as symbolic deixis in explaining the meaning of the word “change”. Such a symbolic deixis relates to the pragmatic usage of semiotics, enabling audiences to understand the meaning of the speaker’s utterance.\n\nThe example of Matt’s use of pictures to present his topic also showed that his purpose of making a speech is to interact with his audiences. Kemi DaSilva-Ibru, a women’s health specialist, gave a topic The Shadow Pandemic of Domestic Violence During COVID-19 on TED. Similarly to Matt, Kemi also offered pictures when she talked to her audiences, but the purpose of using the pictures might be slightly different from Matt.\n\nIn Kemi’s talk, a group of key words constantly appeared such as “pandemic”, “lockdown”, “freedom”, “safety”, and so on. These words are not used to describe the situation of the COVID-19 pandemic, but they relate to the word “violence”. As Kemi introduced in her talk, in the first two weeks of the lockdown in Lagos State, the emergency calls from domestic violence cases were continuous at this time. Kemi shared a photo of a white telephone on the left and a dozen little white lights on the right that were both glowing red, raising the audience’s awareness of violence against women.\n\nThe same themes keep popping up in Kemi’s speech. Since the topic mentions that women in Africa have suffered violence during their quarantine period, audiences certainly look forward to hearing effective solutions to help these women get out of their house. Kemi suggested in her talk that her team successfully “trained 1,300 of the community-based gatekeepers in addressing the cases of violence against women during the lockdown”, offering the necessary assistance to women to receive care. Then a group of young ladies wearing dark blue uniforms can be seen in Kemi’s illustration, standing in the midst of the line while donning masks and projecting an air of authority.\n\nIn this sense, the pictures Kemi posted were consistent with her narrations, while the examples of red lights heralding an increase in violence against women and the building of community-based team heralding the protection of women during the lockdown might aim at illustrating that the speaker’s representation of the content was consistent with the listeners’ perception, resulting in their believing in these representations (Cai, 1997:17-18) of the two related events.\n\n\n5. Conclusion\n\nThis paper primarily attempts to provide readers with a means of analyzing COVID-19-related topics on TED by using the theory of systemic functional linguistics. Specifically, the work analyzed both semantic and semiotic functional usage from several presenters who discussed the theme. In identifying the terms (i.e., “disease”, “medical”) and their relative meanings, along with the presenting of events or activities, functional principles of ideational meaning and interpersonal meaning were particularly manifested.\n\nFirstly, speakers focused on linking semantics with the context, which potentially helped them explain the ideational meaning of the keywords. In other words, speakers were likely to grasp the meta keywords and presented them as compound phrases (i.e., “common cold”, “pulmonary disease”, “Internet of Medical Things”), or short sentences. These phrases or sentences appeared under the contexts which were associated with their logical-based semantic patterns, and in this way, the ideational meaning of the original keywords was expanded (e.g., from “disease” to “transmitted disease”). This expanded meaning signals the fractal of the ideational meaning (Zhao, 2016:642).\n\nSecondly, speech is different from the general text-based content; it contains a multimodal discursive environment, and its language includes both verbal and non-verbal symbols which come together to create a semantic environment. The sample pictures (e.g., frequent phone calls) posted by the speakers were integrated with the ideational meaning of the keywords (e.g., “‘violence against women”), phrases and short sentences mentioned by them, indicating a semantic consistency (Matthiessen, 2007:35) between the pictures and the discursive texts. In this sense, images also formed an expressive layer (Halliday & Matthiessen, 1999:25), constituting a denotative semiotic system to describe, illustrate or summary of the textual meaning.\n\nThirdly, from the above extended understanding of the ideational meaning of COVID-19-based topics, it can be seen that keywords used by the speakers are not equal to their inherent lexical meaning, they contain communicative values of the topics in which specific utterance meanings are generated to reflect the communicative intention of the speakers. This in turn prompts the idea that speakers associated their words and sentences with the COVID-19-based topics as relevant context so that audiences could understand what they were talking about. The ideational meaning of the keywords or phrases which speakers presented was in relevance with their opinions (e.g., “pulmonary disease”) or attitude (e.g., “addressing the cases of violence against women”) to explore their emotional responses, while such emotional responses can also be seen as interpersonal meaning in this sense.\n\nMeanwhile, from the presentations of the COVID-19-based topics on TED, it can be seen that speakers not only share content or ideas but viewpoints and attitudes, and this finding implicates the importance of the logical meta-function in systemic functional linguistics, providing an alternative research method for topics such as analyses of the accountability of the propositions, or the effectiveness of verbal communication.\n\n\nData availability\n\nSource data for this study was taken from the following four TED presentations:\n\n1. David Heymann, What We Do (And Don’t) Know About the Coronavirus (https://www.ted.com/talks/david_heymann_what_we_do_and_don_t_know_about_the_coronavirus)\n\n2. Daniel Kraft, How COVID-19 Transformed the Future of Medicine (https://www.ted.com/talks/daniel_kraft_how_covid_19_transformed_the_future_of_medicine)\n\n3. Matt Walker, 4 Ways the COVID-19 Pandemic Changed the Way We Sleep (https://www.ted.com/talks/matt_walker_4_ways_the_covid_19_pandemic_changed_the_way_we_sleep)\n\n4. Kemi DaSilva-Ibru, The Shadow Pandemic of Domestic Violence During COVID-19 (https://www.ted.com/talks/kemi_dasilva_ibru_the_shadow_pandemic_of_domestic_violence_during_covid_19).",
"appendix": "References\n\nCai Y: Context and Meaning Derivation (in Chinese). Modern Foreign Languages. 1997; 75(1): 15–19.\n\nChang CG: A Social Symbolic Perspective of Systemic Functional Linguistics (in Chinese). Contemporary Foreign Languages Studies. 2012; 3: 18–21+159.\n\nFeng DZ: Construction and Classification of Multimodal Metaphors —— From the System Functional Perspectives (in Chinese). Foreign Languages Research. 2011; 1: 24–29.\n\nGao WY: Metaphorical Analyses of the Modal Verbs Within Scientific and technical Contexts (in Chinese). Journal of Changchun Normal University (Humanities and Social Sciences). 2012; 31(8): 51–52.\n\nGong CH: Studies on Discourse Genre with Systematic Functional Linguistics (in Chinese). Journal of University of Science and Technology Beijing (Social Sciences Edition). 2011; 27(3): 6–12.\n\nGu JM: Contextual and Semantic Studies (in Chinese). Journal of Henan University (Social Sciences). 2004; 44(6): 80–82.\n\nGuan W: Covid-19 Creates the Largest Shock Since World War II (in Chinese). Energy Research & Utilization. 2020; 3: 8–9.\n\nHakibou A: Interpersonal Meaning in Half of a Yellow Sun by Chimamanda Ngozi Adichie through Some Extracts. International Journal of Humanities Social Sciences and Education (IJHSSE). 2022; 9(6): 22–29.\n\nHalliday MAK:Language Structure and Language Function.Lyons J, editor. New Horizons in Linguistics. Penguin;1970; pp.140–165.\n\nHalliday MAK, Matthiessen C: Construing Experience Through Meaning: A Language-based Approach to Cognition. London/New York:Cassell;1999.\n\nHe FZ: Meta-functional Analyses of the Humor in Verbal Communication (in Chinese). Shidai Wenxue. 2009; 2009(8): 105–107.\n\nHong Y, Liang LM: Open Resources from the Elite to the Public: The Rise of TED and Its Implications (in Chinese). Mod. Educ. Technol. 2013; 23(4): 12–15.\n\nLi XY: The Limitations of Systemic Functional Linguistics: The Case of Online Expressions (in Chinese). Journal of Harbin Vocational&Technical College. 2019; 6: 145–147.\n\nLocher MA, Graham SL:Introduction to Interpersonal Pragmatics.Locher MA, Graham SL, editors. Interpersonal Pragmatics. Berlin:Mouton;2010; pp.1–13.\n\nLou Q, Zhang W: Speech Act of “Speaking” and Dual-Order Linguistic Perspective (in Chinese). Foreign Language Research. 2009; 147(2): 74–77.\n\nMatthiessen C:The Multimodal Page: A Systemic Functional Exploration.Royce TD, Bowcher WL, editors. New Directions in the Analysis of Multimodal Discourse. London:Laurence Erlbaum Associations;2007; pp. 1–62.\n\nWang L, Lei YJ: The Focus and Countermeasures of International Discourse Power on Major Public Health Events – Take COVID-19 as an Example (in Chinese). Journalism Lover. 2022; 3: 30–34.\n\nXu LJ: Theories of Linguistic Stratification – From Saussure to Halliday (in Chinese). Journal of Northeastern University (Social Science). 2014; 16(1): 102–106.\n\nYang XQ: The Strata of the Language and the Construction of Systemic Functional linguistics (in Chinese). Foreign Language Research. 2015; 149(1): 24–28.\n\nZhao WC: Fractal of Ideational Meaning in Systems Functional Linguistics (in Chinese). Modern Foreign Languages. 2016; 39(5): 638–646.\n\n\nFootnotes\n\n1 The statement can also be seen from UN chief says COVID-19 is worst crisis since World War II, and is cited by ABCNews (available from https://abcnews.go.com/US/wireStory/chief-covid-19-worst-crisis-world-war-ii-69905340), Fox News (available from https://www.foxnews.com/world/un-coronavirus-challenging-crisis-since-world-war-two ), Africa News (available from https://www.africanews.com/amp/2020/04/01/covid-19-is-worst-global-crisis-since-world-war-ii-un-chief/), and other media, [accessed 12/09/2022]."
}
|
[
{
"id": "192224",
"date": "22 Aug 2023",
"name": "Danang Satria Nugraha",
"expertise": [
"Reviewer Expertise Theoretical Linguistics"
],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 should be a clear purpose/aims, instruments, and major results with global implications highlighted in the Abstract. Kindly add a few more sentences regarding it.\n\nI also expect there is a clear novelty from every article. I did not see it discussed, mentioned, or presented comprehensively in Introduction/Discussion or any sections in the manuscript. You may highlight it at the end of your introduction section for a better display.\n\nThere should be clear aims and wider implications of the study highlighted in the Introduction section.\n\nThere should be clear and justified steps (Theory used) on how to collect (Instrument used), analyze, and interpret data in the Method section. Therefore, it will be easy for another study to replicate it.\n\nThe Discussion should bring new insight and have sufficient citations from the previous studies in the related context. This is a must.\n\nThe Conclusion has not adequately explained the significant contributions of the research and its novelty.\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? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate? Not applicable\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes\n\nIs the case presented with sufficient detail to be useful for teaching or other practitioners? No",
"responses": []
},
{
"id": "203439",
"date": "18 Sep 2023",
"name": "Elih Sutisna Yanto",
"expertise": [
"Reviewer Expertise Systemic functional linguistics in Language education"
],
"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\nOverall, this research cannot provide an interesting topic for readers and language education, and I would like to propose not to approve it: the article is of very poor quality and has fundamental flaws that seriously undermine the methodology, findings and conclusions. All my comments can be seen in the review. Here are some important comments.\nAbstract: The abstract is a brief summary of your paper. I suggest revising the abstract. I suggest the following changes in the organization. Sentence one you would present the topic or focus of your study; in sentence two you would present your study’s research question; in sentence three you would present your study’s documents/participants and methodology; in sentence four you would present your findings; and in sentence five you would present the main points from your discussion of the findings.\nIntroduction:\nIn the introduction, the authors should state their research gap. It is critical for setting the context, justifying the study, engaging the reader, and illustrating the significance and relevance of your research in relation to existing knowledge. It is an essential part of the scholarly communication process and ensures that your research makes a relevant contribution to the academic discourse.\n\nThe paragraph is extremely wordy and contains unnecessary content. The introductory statement, \"Speech has multiple forms and is a type of communication between presenters and audiences,\" for example, might be shortened to \"Speech is a type of communication between presenters and audiences.\"\n\nThe paragraph introduces several concepts such as \"net-language,\" \"scientific vocabularies,\" and \"systemic functional linguistics\" without defining or explaining them. This can leave readers confused about the focus and context of the article.\n\nPhrases such as \"multimodal pragmatic means\" and \"pragmatic functional innovation\" are laden with jargon and may be unclear to readers who are unfamiliar with them. It would be advantageous to offer precise definitions or explanations.\n\nThe paragraph references a number of authors and their works (e.g., Gu, 2004; Li, 2019; Gao, 2012; Feng, 2011; Zhao, 2016; Cai, 1997) without providing context or explaining how these references relate to the topic of the article. It is essential to briefly introduce or explain the relevance of these sources.\n\nThe authors ought to attempt for better clarity, conciseness, and coherence in this paragraph. They should also provide context for the listed references and a clear thesis statement to guide the reader through the main content of the paper.\n\nThe paragraph begins by citing Systemic Functional Linguistics and its inventor, but there is no background provided. It makes no mention of why this linguistic theory is essential or relevant to the research. It would be more informative if some background or context were provided.\n\nMethodology: The methodology used in this study is a case study. However, I do not see the authors explaining the research design clearly and convincingly.\nFindings and discussion: They are not discussed clearly.\nConclusion: The conclusion of the journal article has several weaknesses, including lack of conciseness, unclear articulation of significance, repetition, lack of discussion of limitations, lack of a clear call to action, complexity of language, and lack of specific examples. Addressing these weaknesses would improve the overall clarity and effectiveness of the conclusion.\nFor example:\nThe conclusion is somewhat lengthy and contains multiple complex lines, making it difficult for readers to grasp the essential points. To improve readability, it should be more concise and simpler.\n\nWhile the conclusion mentions the importance of the logical meta-function in systemic functional linguistics, it does not clearly articulate why this analysis is significant or how it contributes to the broader understanding of COVID-19 topics on TED. A more explicit explanation of the study's implications is needed.\n\nThe conclusion does not address any limitations of the study or potential areas for future research. Acknowledging limitations and suggesting directions for future research would add depth to the conclusion.\n\nIs the background of the case’s history and progression described in sufficient detail? No\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? No\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": []
},
{
"id": "203449",
"date": "18 Sep 2023",
"name": "Magdalena Ngongo",
"expertise": [
"Reviewer Expertise Linguistics: Systemic Functional linguistics Approach"
],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nTechnically, there are five paragraphs which consist of only one compound or complex sentences. I suggest to combine them in a paragraph (see point 4.1)\n\nThe view of ideational and interpersonal meaning should be added based on available data. Especially relating to the transitivity (i.e. process types are used by speakers) to express his/her idea. So does the interpersonal meaning (i.e. which types mood clause is used) to exchange experience (point 4.1).\n\nIf it is possible, add more examples and explanation relating to Semiotic covering the 4 data of texts in order to make it complete (point 4.2).\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? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate? Yes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes\n\nIs the case presented with sufficient detail to be useful for teaching or other practitioners? Partly",
"responses": []
},
{
"id": "203445",
"date": "25 Sep 2023",
"name": "Loretta Fernandez",
"expertise": [
"Reviewer Expertise Applied",
"linguistics",
"education",
"Systemic Functional Linguistics",
"Sociocultural Theory"
],
"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 addresses an interesting and worthwhile topic in a very limited and superficial way. The article analyzed (with an analytic method not explained) some instances of language in TED (Technology, Entertainment, and Design) talks about COVID 19, using a vague notion of Systemic functional Linguistics (SFL). Systemic Functional Linguistics is an extremely complex and ramified theory of language. The authors did not use any of the seminal works (e.g., Halliday and Matthiesen, 2014; Thompson, 2013; Eggins, 2004) in which are explained the principles of SFL analysis, that would have helped them understand in depth the complexities of the word ‘functional’ in the theory. Moreover, the authors claimed they wanted to ‘reintrerpret’ SFL, but to reinterpret a theory you have first to be able to present it and the case studies cited in the review of literature are not enough and not representative of the type of analysis the authors proposed. Fore example, the authors might have used of the notions of Register and Genre (e.g., Martin & Rose, 2012) or multimodal analysis of images (Kress & vanLeuween, 2021) to interpret the TED talks about COVID 19. The TED talks as genres could have been analyzed in their stages and analyzed at the lexical grammatical and semantical levels highlighting the ideational and interpersonal metafunctions. But for examples the authors talk about Themes (p. 7), which are typical of the textual metafunction in an inadequate way. It would have been more useful to do one analysis following a sound methodology whether metafunctional (e.g., Eggins, 2004) or genre based (e.g., Martin and Rose, 2007) than the intuitive analysis that the authors of this article present that confuses and mixes together different types of SFL analyses and multimodal analyses.\n\nThe article begins highlighting the importance of the TED talks and how the topic of COVID-19 is particularly relevant in this historical moment. They argue that there are relatively few studies that address SFL analyses of TED talk and that it would be useful to do so as this is a very important genre of communication. In my view, this hypothesis is the only strong point of the article. Unfortunately, the authors do not demonstrate a deep enough knowledge of SFL to perform the type of analysis they would have needed to prove their hypothesis. The article also would benefit from some revisions on the use of the English language, in particular of the articles he/she/they, etc. which are often used in different ways for the same person. Another aspect I would address is that the authors refer to the TED talk speakers by first name instead of last name with is a convention of academic English and makes it also easier to cite in the bibliography which does not present all the works cited.\nTo conclude, I think that the claims made in the discussion and conclusion are not sustained by the data and data analysis presented in the article. The initial idea of the authors is worth researching but they have to do a much more rigorous investigation and use a specific SFL methodology to do the analysis and highlight the lexical grammatical and semantic aspects they wanted to highlight.\nReferences Eggins, S. (2004). Introduction to Systemic Functional Linguistics. London- NY, NY: Continuum. Halliday, M. A. K. And Matthiessen C. M. I. M. (2014). Halliday’s Introduction to Functional Grammar. London: Routledge. Kress, G. and Van Leeuwen, T. (2021). Reading Images: The Grammar of Visual Design. London: Routledge Martin, J. R. & Rose, D. (2007) Working with Discourse Meaning Beyond the Clause. New York: Continuum. Thompson, G. (2013). Introducing Functional Grammar (3rd ed.). Routledge. Https://doi.org/10.4324/9780203431474\n\nIs the background of the case’s history and progression described in sufficient detail? No\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? Not applicable\n\nAre all the source data underlying the results available to ensure full reproducibility? No\n\nAre the conclusions drawn adequately supported by the results? No\n\nIs the case presented with sufficient detail to be useful for teaching or other practitioners? No",
"responses": []
}
] | 1
|
https://f1000research.com/articles/11-1515
|
https://f1000research.com/articles/11-1514/v1
|
13 Dec 22
|
{
"type": "Study Protocol",
"title": "Community-based case studies of vaccine hesitancy and the COVID-19 response in South Africa - study protocol",
"authors": [
"Charles Shey Wiysonge",
"Nancy Coulson",
"Nirvana Pillay",
"Sara Cooper",
"Candice Groenewald",
"Zaynab Essack",
"Saahier Parker",
"Gregory Houston",
"Jane Simmonds",
"Anelisa Jaca",
"Muyunda Mutemwa",
"Patrick DMC Katoto",
"Heidi van Rooyen",
"Nancy Coulson",
"Nirvana Pillay",
"Sara Cooper",
"Candice Groenewald",
"Zaynab Essack",
"Saahier Parker",
"Gregory Houston",
"Jane Simmonds",
"Anelisa Jaca",
"Muyunda Mutemwa",
"Patrick DMC Katoto",
"Heidi van Rooyen"
],
"abstract": "Background: In 2021 the South African government launched a large COVID-19 immunization campaign with the goal of reaching more than 40 million individuals. Nonetheless, certain international largely internet-based surveys at the time showed a significant proportion of vaccine hesitancy in South Africa. This study aims to determine and co-create with local stakeholders a comprehensive understanding of vaccine hesitancy and opportunities to support the promotion of other COVID-19 health-promoting behaviours at community level. Methods: A mixed-methods multiple case-study design; informed by the socio-ecological model of behaviour change. Four socio-economically diverse communities across South Africa will be selected and data collection will take place concurrently through three iterative phases. Phase 1 will provide insights into community experiences of COVID-19 (response) through desktop mapping exercises, observations, in-depth interviews, and focus group discussions (FGDs) designed as expression sessions with local stakeholders. Phase 2 will explore the extent and drivers of community acceptance of COVID-19 vaccines. This phase will comprise a survey based on WHO’s Behavioural and Social Drivers of Vaccination tool as well as further FGDs with community members. Phase 3 will involve cross-case study syntheses and presentation of findings to national role-players. Discussion: This study will provide ground up, locally responsive, and timeous evidence on the factors influencing COVID-19 health-seeking behaviours to inform ongoing management and mitigation of COVID-19 in South Africa. It will also provide insights into the applicability of a novel vaccine hesitancy model in Africa.",
"keywords": [
"South Africa",
"COVID-19 response",
"vaccine hesitancy",
"case studies",
"behavioural and social drivers of vaccination",
"non-pharmaceutical interventions"
],
"content": "Introduction\n\nSouth Africa first went into national lockdown on 26 March 2020 in response to the coronavirus disease 2019 (COVID-19) pandemic and has since aligned with international guidance led by the World Health Organization (WHO) on how to manage the unprecedented pandemic in the face of emergent scientific evidence.1,2 Lockdown regulations implemented in varying degrees by the South African government responded to the waves of COVID-19 amidst increasing uncertainty and social and structural damage to South Africa.3 During the first quarter of 2021, the country experienced serious levels of COVID-19 infections as the second wave of COVID-19 infections unfolded across the country.4 Internationally and locally, new variants of COVID-19 were found to be more infectious and the surge in COVID-19 cases at the beginning of 2021 was more serious than the infection levels experienced at the start of the pandemic in 2020.5\n\nThe scientific pursuit of biomedical interventions to manage COVID-19 saw the proliferation of studies on drug and vaccine efficacy during 2020.6 Over the year, the need to manage and translate the changing landscape of COVID-19 evidence as it emerged was ever-present. This complicated the implementation and maintenance of behavioural interventions regarding non-pharmaceutical interventions (NPIs), but also emphasised the need for NPIs given we had nothing else at the time. In early 2021, NPIs were the only available and accessible methods to reduce and prevent the spread of COVID-19 in the country.1,2 At the same time, the safety and efficacy of a number of COVID-19 vaccines had been established and the manufacture, procurement, and rollout of these vaccines was a global priority.7 Contrary to high-income countries, at the beginning of 2021, limited numbers of vaccine doses had been procured for South Africa and ongoing procurement and rollout was uncertain.8 However, vaccinating the South African population at scale with efficiency was a top government priority.9 COVID-19 vaccine uptake will involve behaviour change at a large scale as the government aims for a vaccination coverage of at least 67% of the South African population in order to control COVID-19 infections in the country.\n\nVaccine hesitancy is one of the main challenges to be addressed if the vaccine programme is to be successful in South Africa.10 Vaccine hesitancy, according to WHO’s global working group on “Measuring Behavioural and Social Drivers of Vaccination” is a “motivational state of being conflicted about, or opposed to, getting vaccinated” which includes intentions.11,12 Limited evidence available in early 2021 showed wide variation in COVID-19 vaccine hesitancy from 18% to 48% across various time points and geographical locations in South Africa.13,14 Many elements could have contributed to the vaccine hesitancy. For example, previous experiences that communities have of the delivery of healthcare services could influence both vaccine intentions and uptake.15 Contextual factors that shape the enabling environment for vaccine uptake within a middle-income country like South Africa might be expected to play a bigger role in levels of vaccine hesitancy when compared with the results found in other studies which are largely conducted in high-income countries.16,17 For example, the availability and cost of transport is regularly a barrier to access to healthcare services in resource constrained communities.18,19 In addition, the dominance of social media has been shown in a global study to directly impact on vaccine uptake.20 Even before the COVID-19 pandemic, South Africans were increasingly finding themselves in “echo chambers” of misinformation about vaccination on social media and the internet.21–27\n\nEnsuring optimal uptake of COVID-19 vaccines in South Africa would thus involve multiple factors including knowledge, creating an enabling environment, addressing social influences, and personal motivation.19,28,29 The preceding paragraphs highlight the need for a ground up, contextually informed approach, that recognises the lived experience and understanding of individuals and places them in the social world in which they live and work. That was the rationale for the proposed study, which aims to determine and co-create with local stakeholders in four diverse South African communities, a comprehensive understanding of vaccine hesitancy and opportunities to support the promotion of COVID-19 health seeking behaviours.\n\nThe specific objectives of the proposed study are to:\n\n1. Create a community-based COVID-19 relevant geographic information system (GIS) picture that can be used for local planning in the COVID-19 response, including for vaccination and NPIs to curb the spread of COVID-19.\n\n2. Document stories of the lives of community members affected by COVID-19 sickness, death, and economic loss in four communities.\n\n3. Use the socio-ecological model to provide a community level assessment of the barriers and enabling factors at various levels (personal, interpersonal, community, and social/political) to vaccine acceptance and for ongoing adherence to behavioural measures.\n\n4. Pilot the behavioural and social drivers of vaccination (BeSD) survey tool to provide guidance on its application in South Africa and to baseline vaccine hesitancy at the local level.\n\n5. Co-create with community-level stakeholders, context sensitive strategies to address vaccine hesitancy and COVID-19 health seeking behaviours to guide both local and national responses.\n\n\nMethods\n\nFour community-based case studies will be undertaken using the socio-ecological model.30 The research methods to be applied in the case studies are described in further detail in the following sections. Each component of data collection will be complemented by a process of engagement with stakeholders at the community level, described as action research.31 These stakeholders will be identified during community-level mapping, and will include local community leadership and service providers, such as health facility managers and school principals. These and other key informants will be interviewed and then invited to participate in workshops to contribute to the interpretation of findings and to co-create community-level strategies to address vaccine hesitancy and to strengthen the COVID-19 response as findings become available.\n\nWe propose using the socio-ecological model for behaviour change as a framework to understand how to increase COVID-19 vaccine uptake by looking at barriers and enabling factors at various levels, ranging from the individual to the interpersonal (the influence of family and peers) through the community to the wider context (policy, infrastructure, and resources). Health messaging and correct information, although important, is often not enough for people to implement sustainable behaviour change. The socio-ecological model acknowledges that for any behaviour change to be maintained and sustained, it needs to happen at many different levels. Information about COVID-19 vaccines in isolation to other interventions will not be sufficient to get the uptake required for herd immunity; information and strategies that address vaccine hesitancy and that motivate people to access COVID-19 vaccines are also needed. It is critical to behaviour change in a community that all four levels — personal, interpersonal, community, and social/political — be capacitated for a change in behaviour to occur in many people and then be maintained and sustained. Box 1 illustrates how the different levels of the socio-ecological model can be applied to address vaccine hesitancy. We combined our knowledge of the socio-ecological model of behaviour change as well as vaccine hesitancy to prepare this box. In practice, we have contextualised tools recently developed by the BeSD working group15 and used them in this study. The BeSD framework consists of four domains that could potentially shape vaccine intentions and uptake, namely, what people “think and feel about vaccines; social processes that drive or inhibit vaccination; individual motivations (or hesitancy) to seek vaccination; and practical factors that shape the experience of seeking and receiving vaccination.12\n\nIndividual (Self): understand the information about the vaccine (know that it is available and it is recommended that you get it), be motivated to get it, and be able to get access.\n\nInterpersonal: getting the vaccine needs support from partners, family members, and peers, or at least encouragement for this behaviour.\n\nCommunity: getting the vaccine needs to become a social norm and the socially accepted and expected thing to do. You need to be able to access the vaccine easily and safely at a local primary healthcare facility, doctor or workplace. Religious organisations, community organisations, local leaders, traditional leaders, traditional healers, and other influencers (social and political) need to be supportive of vaccine uptake.\n\nEnvironment: At a public policy level, the availability of the vaccine needs to be secured and the vaccine needs to be available. Resources need to be available to place vaccination centres within easy reach of the community.\n\nThe BeSD tools include qualitative tools (guides for in-depth interviews with stakeholders), quantitative tools (questionnaires for population surveys), and a guidebook to support implementation of the tools. The tools, which focus on childhood vaccination and COVID-19 vaccination for health workers and for adults, are publicly available from the WHO publications cited in this sentence.12,15 We have adapted the BeSD COVID-19 vaccination tools for adults to our local context.32 Using the collective expertise and experiences of the study team, we adapted the tools by removing items that are not applicable to the South African context and adding items that we deemed were missing from the tools.\n\nThis research is designed as multiple case studies of four sites. Case study research is empirical research that explores a contemporary phenomenon within its real-world context.33 Case studies are especially useful when the boundaries and interface between the “phenomenon” (or “the case”) and the “context” are not clear.33 Case studies usually rely on multiple sources of data.33 In our study, the “phenomenon” is the introduction of COVID-19 vaccination into the four selected communities in South Africa with their different contextual factors, histories, and experience of COVID-19. Case studies are dominated by ‘how and why’ research questions.33 By adopting an approach that relies on more than one data collection method, it is possible through a case study to describe, explore, and explain the “how and why” of possible vaccine hesitancy without presuming at the outset to know the contextual and other factors that are shaping local behaviour. The research methods will be carefully replicated at each site; the underlying goal being either that the research will provide the replication of results across sites or that the case studies will provide theory that can predict either similar or contrasting findings across different communities.33 In either case, multiple case studies will provide evidence that will be helpful to the activities of the South African Government in addressing vaccine hesitancy.\n\nTable 1 shows the site selection of the four case studies. Convenience and purposive sampling were used in the selection of sites. Convenience sampling ensured that some sites which are well known to the study team with established research and community networks to facilitate speedy data collection can be used. Purposive sampling will allow the team to include a mix of sites from different South African provinces badly affected by COVID-19 and to include communities that reflect formal and informal urban contexts as well as peri-urban and rural environments. Study sites were selected in KwaZulu Natal, Gauteng, and Western Cape provinces, which were the three provinces with highest numbers of COVID-19 cases in South Africa in early 2021.34 In KwaZulu Natal Province we selected an urban (Wentworth) and a semi-rural community (Sweetwaters). In Gauteng Province we selected an urban informal community known as Alexandra. Finally, in the Western Cape Province, an urban middle-class community was selected. The selected sites have markedly different populations as shown in Table 1.\n\n* The source of the provincial COVID-19 data is the South African National Department of Health, as reported on 29 January 2021.34\n\nAt each case study site, teams will use a mixed methods approach to data collection with quantitative and qualitative elements to address all research objectives. Figure 1 illustrates the overall approach to the study that is to be replicated in each of the case study sites. At each site data collection and the dissemination of findings will be organised into three distinctive phases.\n\n(BeSD, behavioural and social drivers of vaccination; KIIs, key informant interviews; FGDs, focus group discussions; GIS, geographic information system; MAC, ministerial advisory committees)\n\nPhase 1 will provide a preliminary snapshot of the community experiences of COVID-19 and the responses to it. This will be achieved using GIS maps to visualise the spatial arrangements for infrastructure, housing, employment, and public and private services, as well as population centres and COVID-19 statistics. Thereafter key informant interviews (KIIs) with community stakeholders and focus group discussions (FGDs) designed as expression sessions will provide a narrative about the COVID-19 experience in the local community. The local map and narrative will be shared with key informants at a workshop where the findings will provide the basis for the selection of population groups for a deeper investigation of vaccine hesitancy in phase 2.\n\nIn Phase 2, the case study research design will throw a spotlight on community acceptance of COVID-19 vaccines. A quantitative survey and further FGDs will be conducted with groups in the community. The findings of the survey and focus group discussions will be shared with the key informants at a second workshop, and this will form the basis for stakeholders co-creating strategies to maximise vaccine acceptance within their ward.\n\nPhase 3 will involve cross case study syntheses as appropriate, and the presentation of findings to national role-players including the National Department of Health (NDoH), the Ministerial Advisory Committee (MAC) on COVID-19 Vaccines, the MAC on COVID-19, and the Behaviour Change Communication MAC. In Phase 3 researchers will prepare an integrated report across all four case study sites. Academic papers will be prepared and complemented by a comprehensive literature review of vaccine hesitancy and behavioural interventions as secifically experienced in Africa and in other cases where local perspectives about vaccine acceptability are integrated into national vaccine programmes.\n\nTable 2 summarises the study population for both the qualitative and quantitative components. The study will only recruit people who are aged 18 years or older.\n\nStudy population for qualitative component\n\nDuring Phase 1 we will conduct 15-20 key informant interviews (KIIs) with stakeholders who are purposively selected. The proposed list of ward-based key informants includes but is not limited to local political and traditional leadership, clinic and health service leaders, school principals, faith-based leaders, business leaders, government officials, local taxi associations, local civic leaders, local trade union leadership, and private healthcare practitioners. It is expected that key informants at each site will include many of the individuals listed above, but also that at each site a unique list of key informants will emerge iteratively. During the interviewing of key stakeholders, researchers will enquire after the names of other potential key informants to ensure that influential leadership at the local level is not inadvertently overlooked during the mapping exercise. Snowball sampling would ensure that the research team identifies all significant individuals shaping the local COVID-19 response who may, for example, be outside of the immediate community such as provincial and/or national role-players.\n\nParticipants for both types of FGDs will be purposively identified. Three FGDs of each type will be held at each site. During Phase 1 no more than six participants will be recruited into the expression sessions. The sessions will be implemented in a group setting consisting of no more than six participants. The small sample size here is deliberate, to allow for meaningful engagement in the focus group discussions. Participants who have been badly affected by COVID-19 either through sickness, death, or economic loss will be recruited into the expression sessions. A social worker will be available at each site to ensure that any trauma experienced during or after the sessions can be appropriately referred. In Phase 2, between six and eight participants will be purposively recruited into the FGDs that are to be supported with a semi-structured interview guide. Specifically, this group of respondents are those best placed to influence social norms around vaccination in the community. The participants for these FGDs will be identified at the end of Phase 1 and in discussion with community stakeholders. This could include healthcare workers, faith-based leaders, community leadership, and other community and social workers.\n\nStudy population for the quantitative component\n\nThe selection of survey respondents will be guided by priority populations in line with the rollout phases and target groups for COVID-19 vaccination as determined by the NDoH. The findings of Phase 1 research and consultation with local stakeholders will also determine the final selection of the survey respondents. It is expected that it may include priority groups at risk of COVID-19 (the elderly), essential service workers identified in each study site (for example, teachers, taxi drivers, and people working in institutional care facilities) and other adult men and women in the selected communities. The participants should also be resident or working in the community where the survey is to be conducted. The study will use non-probability convenience sampling because it is a cost- and time- effective method. It is proposed to collect 300 completed survey questionnaires at each site. A sample comprising at least 300 participants per site allows for the detection of small correlations (r=0.2) with at least 95% power.35\n\nThree discrete but interlinking components of data collection will be undertaken for each case study site. These are: desk review of demographic and epidemiological profiles and available GIS mapping and literature; qualitative data collection methods; and quantitative data collection using a questionnaire.\n\nDesk based methods\n\nA desktop mapping exercise using existing GIS and COVID- 19 related documents will complement qualitative and quantitative data collection at each site. The desk review will aim to create a comprehensive picture of each community. This will include a demographic and epidemiological profile, and mapping of the local community using general spatial planning data commonly stored on GIS and the collection of documents that describe the COVID-19 response in the community. A COVID-19 relevant picture of each community will be created, using GIS to support this, and will include: COVID-19 epidemiological profile; population (numbers, priority and at-risk groups, and population centres); services and local infrastructure (schools and, clinics); transport routes (taxis and buses); civil society (faith-based organisations, women groups, and non-governmental organisations); government offices (mayor’s office and relevant departments, police, etc); private sector (large employers and trade union representatives, private health care); and COVID-19 response (who has done what, where, and the size of the spend). The desk-based mapping and evidence review will inform the sampling for the key informant interviews with stakeholders described later.\n\nQualitative methods\n\nKey informant interviews\n\nThe mapping exercise will identify key stakeholders in each community to be approached for interview. Researchers will use a semi-structured interview guide, adapted from the BeSD qualitative tools for COVID-19 vaccination, for KIIs. The latter will be conducted face-to-face at the community level or over virtual platforms; depending on COVID-19 conditions at the time, participant preferences, and other practical considerations. The KIIs will explore a range of themes including: participants’ personal, family and community experiences of COVID-19; the impact of COVID-19 on personal and work life; experience of varying lockdown regulations; experience of adherence to NPIs; understanding of COVID-19 vaccines; how COVID-19 has shaped their leadership role in the community; and expectations for the future. All research teams will consist of a mix of local language and English-speaking researchers. With permission of participants, interviews will be audio-recorded and thereafter translated as necessary and transcribed.\n\nObservation\n\nOn-site researchers will conduct observations of community life in the case study sites. These observations will be centred around locations where community members meet, such as shopping centres, street trading spaces, and/or taxi ranks. The purpose of the observations will be to better understand local community life as well as to document observation about NPI adherence as well as COVID-19 related media and facilities, such as for hand washing.\n\nFocus group discussions\n\nTwo types of FGDs will be conducted. The first type will apply an approach called “expression sessions”.36 The second type of FDG will rely on a semi-structured FGD guides. Expression sessions will be conducted in Phase 1 of the research activities. The second type of FGD will follow in Phase 2. All FGDs will be conducted face-to-face at the research site and will follow COVID-19 precautions including physical distancing, the wearing of face masks, and a well-ventilated venue. FGDs will be capped at six participants for expression sessions and eight for other FGDs. The tools will be piloted in one pre-selected community and amendments made thereafter. All FGDs will be audio recorded with the permission of participants. Thereafter recordings will be translated as necessary and transcribed.\n\n“Expression session” focus group discussions: Expression sessions will be facilitated with participants in the community who have been severely affected by COVID-19 through personal ill-health, family loss, and/or economic adversity. An expression session is a newly developed approach to collect verbal, visual, and audio-visual data from participants.37 This approach is informed by the photovoice methodology where participants are required to take photos in response to research questions.38 The approach encourages active participation in research, and meaningful reflection and engagement with research questions, which is important in the context of COVID-19 and vaccine hesitancy. Expression sessions encourage participants to respond to probes by sharing ‘something’ rather than photos only. ‘Something’ could be images, photos, videos (self-taken, downloaded online, or from a book or magazine), a song (audio or lyrics), a drawing (self-drawn or a picture of a drawing), or a poem (self-written or not). Participants will be encouraged to bring their ‘somethings’ to an FGD. Participants will receive a brief formative training session on the methodology along with a set of two research probes to which they can respond by bringing ‘something’ (see [47]). Following the formative session, participants will have one week to gather their ‘somethings’. Expression session FGDs will be facilitated by two researchers and will be conducted in the dominant or preferred language of the group. Each team will include a researcher with local language capacity. Participant information sheets and consent forms will be translated into the local language.\n\nFocus group discussions with semi-structured guides: Conventional FGDs will be conducted with three pre-defined stakeholder groups who are able to address vaccine hesitancy in the community and provide support to the local response to improve acceptance and uptake of vaccines. These FGDs will focus on how to strengthen the community response to vaccine hesitancy and to promote on-going COVID health seeking behaviours. The procedure for the semi-structured FGDs is found in [47]. FGDs will be facilitated by two researchers. It is anticipated that most of these FGDs can be conducted in English but researchers with local language capacity will be included in each research team. Participant information sheets and consent forms will be translated into the local language.\n\nQuantitative methods\n\nFor the quantitative component of the study, we adapted the BeSD quantitative COVID-19 vaccination tool for adults.15 The tool ([47]) consists of a questionnaire that assesses four domains i.e., what adults think and feel about COVID-19 vaccines; social processes that drive or inhibit COVID-19 vaccination; individual motivations (or hesitancy) to seek COVID-19 vaccination; and practical factors that shape the experience of seeking and receiving COVID-19 vaccination.15 The survey will be conducted in English, Afrikaans, and isiZulu since these are predominant languages spoken in the selected communities. Participant information sheets and consent forms (see [47]) will also be translated into the local language. The survey tool will be piloted among 100 adults in one of the first community sites. Where necessary, changes will be made to the survey to assist with the translation of specific terms. Data will be captured onto tablets during face-to-face interviews conducted by well-trained researchers working in each community site.\n\nCOVID-19 precautions\n\nThe research team will adopt an approach to data collection that is compliant with all the rules pertaining to the COVID-19 response. The researchers will wear masks, practice physical distancing when approaching potential participants, use hand sanitisers regularly, and adhere to all other COVID-19 protocols. Should the period for interviews in any given community coincide with the period of lockdown, face-to-face interviews will be replaced with online questionnaires. It is expected that some qualitative data collection will be virtual and meetings to discuss findings with local stakeholders may be virtual where appropriate. Onsite researchers will use personal protective equipment (PPE, including face masks and hand sanitiser) and will provide PPE to FGD participants. Meetings and FGDs will be made in well ventilated venues and seating will be appropriately physically distanced.\n\nIterative analyses of all data will occur at each community site as the research team moves from Phase 1 to Phase 3. Data will be triangulated across methods to promote validity. Case-specific and cross-case syntheses will be undertaken as appropriate and as determined by the findings.\n\nQualitative data analyses\n\nQualitative data will be audio recorded, transcribed, and translated where required. Transcripts will be organised and stored using qualitative data software such as MAXQDA (VERBI GmbH, 2022) and ATLAS.ti (version 9) to facilitate coding and analyses of data. Qualitative data will be subject to thematic analysis.39 A code book will be developed for the data including both inductive and deductive codes,40 but emphasis will be placed on allowing the data to determine codes.41 The codes will be discussed between the four qualitative researchers involved in the case studies and with the four study principal investigators. An inter-coder agreement will be established with a portion of the data to verify and enhance data credibility.\n\nQuantitative data analyses\n\nQuantitative data will be cleaned and stored in the Statistical Package for Social Sciences software, version 27.0 (IBM SPSS Inc, Chicago, IL) (RRID: SCR_016479). Thereafter both the SPSS version 27.0 and R/Rstudio v3 (RStudio, 2022) (RRID: SCR_000432) will be used for data analyses. The survey data will be summarised as counts and percentages for categorical variables and means with their standard deviations for continuous variables. Analysis of the variance (ANOVA), chi-square tests, and equivalents will be used as appropriate for group comparisons. Logistic regression models will also be used to evaluate the association between selected characteristics and vaccine hesitancy as well as adherence to NPIs. A basic model will be adjusted for age and sex. Expanded multivariable models for the outcome “vaccine hesitancy” will be further adjusted for significant predictors in basic models. Additional data analyses will include subgroup analyses conducted using variables such as race, sex, socio-economic status, geographical location, education levels, and occupation to match areas specific needs. A P-value less than 0.05 will be used to indicate statistically significant results.\n\nThis study proposal was approved by the Human Research Ethics Committees of the University of the Witwatersrand in Johannesburg, South Africa (reference: H21/02/05) on 25 March 2021; the Human Sciences Research Council of South Africa (reference: REC 12/04/21) on 20 April 2021; and the South African Medical Research Council (reference: EC022-5/2021) on 27 May 2021.\n\nEvery effort will be made to ensure that the research process is socially sensitive and respects the rights of all participants. Information sheets will be provided to all potential participants (as hard or electronical copies as the case may be) as well as verbal briefing on the nature and purpose of the interviews or FGDs, emphasising the right of individuals to refuse or withdraw from the interview or FGD at any point. A consent form for participation and for audio-recording will be provided. All participation is voluntary. Participants at face-to-face FGDs will be provided with refreshments.\n\nPersonal identifiers during KIIs and FGDs will not be used in the reporting and all data will be reported in a summarised form (sex, age) without attributing comments or information to individuals. FGD participants will be informed that while all steps will be taken towards maintaining confidentiality and anonymity, this cannot be guaranteed due to the nature of sharing encouraged by FGDs. While FGD participants will know each other’s identity and the information shared, an environment of private sharing will be encouraged; highlighting the importance of maintaining confidentiality for all participants. Furthermore, a local social worker will be available at each site to support or refer any participant that experiences any psychosocial concerns during or after the expression session FGDs. All data will only be used for research purposes and kept with utmost confidentiality, and they will not be accessed by anyone else but the research team.\n\nRespondents will be reimbursed for their participation in the study. This is ethically sound and is aligned to the “time, inconvenience, and expenses” (TIE) model42 and is endorsed by the National Health Research Ethics Council of South Africa.43 The reimbursement amounts proposed for the study are aligned to TIE that reimburses participants for their time at a rate on par with unskilled labour rates, for their inconvenience and for any expenses (e.g., transport, childcare). The reimbursement for KIIs and FGDs is ZAR 150 (South African Rands) and for the survey is ZAR 100.\n\nThe study team is multidisciplinary, consisting of specialists in public health, epidemiology, social science, vaccinology, psychology, political science, project management, and health promotion. The team is led by four principal investigators from the three South African institutions, namely, the Human Sciences Research Council (H.v.R), the Sarraounia Public Health Trust (N.C. and N.P.), and the South African Medical Research Council (C.S.W.).\n\n\nDiscussion\n\nPrevious studies have shown high levels of vaccine hesitancy in South Africa.9,14,44 These high levels of are most likely to be driven by multiple factors including personal, interpersonal, community, and social. We therefore propose in this study to determine and co-create with community stakeholders a comprehensive understanding of vaccine hesitancy and opportunities to support the promotion of COVID-19 health seeking behaviours in South Africa. The conceptual framework for the study is based on the socio-ecological model of behaviour change and the BeSD framework for COVID-19 vaccination.15\n\nExisting vaccine hesitancy measurement tools were mostly designed for use in and validated in high-income countries.10,16 Given that vaccine hesitancy is context specific, there is a need to adapt and validate existing COVID-19 vaccine hesitancy tools for use at national and community levels in South Africa. The BeSD tools, which were recently developed under the auspices of WHO, measure both behavioural and social drivers of vaccination.12,15 At the time this study was conceived in early 2021, the BeSD model of vaccine hesitancy had not been validated in an African setting. The proposed study will therefore provide insights into the applicability of a novel vaccine hesitancy model in Africa.\n\nThe backdrop to the proposed community-based case studies is that the benefits of models of behaviour change that are focused purely on knowledge deficit are limited. Understanding both the behavioural (individual) and social drivers of vaccine hesitancy is critical to increasing COVID-19 vaccine uptake in South Africa. The study applies the socio-ecological model to the investigation of vaccine hesitancy. This model considers the complex interplay between individual, relationship (interpersonal), community, and social factors. It allows us to explore the range of factors that render people conflicted about or opposed to COVID-19 vaccination. Although all levels of the socio-ecological model are relevant for communities, researchers and practitioners in health promotion are often poor at articulating the integration of these levels and how this determines effective health promotion strategies. One of the most effective applications of the socio-ecological model is at the community level.30 This is because the enablers or barriers to behaviour change in the four levels (i.e., personal, interpersonal, community, social) of the model can be readily identified.19 For example, if a local faith leader is speaking out against vaccination, it will most likely have an impact on the social norms of the local community.19 Similarly, if the local clinic has a reputation for providing poor services, then this too will affect vaccine uptake because it is a barrier to establishing an enabling environment. A bottom-up approach to understanding vaccine hesitancy provides the best opportunity to understand those factors that encourage or impede vaccine uptake. This is enhanced when community stakeholders are actively engaged in providing an interpretation of the intersection of the range of factors at the community level.\n\nThis mixed methods study is designed as an action research study that is responsive to the local context and is conducted to influence the ongoing management and mitigation of COVID-19 in South Africa. To address the issue of COVID-19 vaccine uptake, an integrated analysis is necessary to provide community stakeholders with a comprehensive understanding of the factors that will assist the identification of helpful health promotion activities at the local level to support COVID-19 vaccine rollout. Convenience sampling is best suited for this type of study, although this would limit the generalisability of the findings to all communities in South Africa. However, we believe that the study will make a substantial contribution to knowledge on COVID-19 response in South Africa, as the case study research will be conducted in four commonplace settings in the country.\n\nWe plan to disseminate the findings of this study through meetings with relevant national stakeholders in South Africa as well as peer-reviewed publications. The collected data will be available on request from the corresponding author.\n\n\nConclusions\n\nThe COVID-19 pandemic continues to have significant health, human, social, and economic impacts on South African society. COVID-19 vaccines and non-pharmaceutical interventions (NPIs) currently offer the most promising means to manage the pandemic. However, their success depends on high levels of uptake and adherence. The aim of this study is to determine and co-create with local stakeholders a comprehensive understanding of vaccine hesitancy and opportunities to support the promotion of other COVID-19 health-promoting behaviours. The study will utilise a mixed-methods, multiple case study design, informed by the socio-ecological model of behaviour change. The study will provide ground-up, locally responsive, and timeous evidence on the factors influencing COVID-19 vaccine acceptance and other health-seeking behaviours to inform the management and mitigation of the pandemic in South Africa. It will also provide insights into the applicability of various global vaccine hesitancy models and research tools to a middle-income country in Africa.\n\nWe have completed data collection for phases one and two and are currently doing analyses. Phase three began in August 2021 and is ongoing. We have published a peer-reviewed paper on the survey component of phase 2.45 The questionnaires used as part of this study are available underthe terms of the Creative Commons Zero \"No rights reserved\" data waiver (CC0 1.0 Public domain dedication) with DOI: 10.6084/m9.figshare.21548826. We also deposited an earlier version of the study proposal in a preprint server.46",
"appendix": "Data availability\n\nFigshare: Questionnaires for the \"Community -based case studies of vaccine hesitancy and the COVID-19 response in South Africa\" https://doi.org/10.6084/m9.figshare.21548826.v1. 47\n\nThis project contains the following extended data:\n\n- Data_BeSD_in_RSA_Wiysonge_14nov2022.docx (All interview, focus group and questionnaire tools)\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).\n\n\nReferences\n\nAbdool Karim SS: The South African Response to the Pandemic. N. Engl. J. Med. 2020; 382(24): e95. PubMed Abstract | Publisher Full Text | Free Full Text\n\nWiysonge CS: South Africa’s War on COVID-19. Think Global Health;20 April 2020. (accessed 22 December 2021). 2020.Reference Source\n\nNDOH-a: National Department of Health, South Africa. Regulations and Relief.2021 [cited 2022 14 January 2022].Reference Source\n\nNDOH-b: National Department of Health, South Africa. Covid-19 Daily Updates & Cases.2021 [cited 2022 14 January 2022].Reference Source\n\nAbdool Karim SS, de Oliveira T : New SARS-CoV-2 Variants - Clinical, Public Health, and Vaccine Implications. N. Engl. J. Med. 2021; 384(19): 1866–1868. PubMed Abstract | Publisher Full Text | Free Full Text\n\nBoutron I, et al.: The COVID-NMA Project: Building an Evidence Ecosystem for the COVID-19 Pandemic. Ann. Intern. Med. 2020; 173(12): 1015–1017. PubMed Abstract | Publisher Full Text | Free Full Text\n\nNdwandwe D, Wiysonge CS: COVID-19 vaccines. Curr. Opin. Immunol. 2021; 71: 111–116.\n\nSisonke: Sisonke study: a pragmatic real world phase 3b clinical trial of the single-dose COVID-19 vaccine candidate among frontline healthcare workers in South Africa.2021. (accessed 19 September 2021).Reference Source\n\nNDOH-c: National Department of Health, South Africa. Vaccine updates.2021 [cited 2022 14 January 2022]. Reference Source\n\nWiysonge CS, et al.: Vaccine hesitancy in the era of COVID-19: could lessons from the past help in divining the future? Hum. Vaccin. Immunother. 2021; 1–3.\n\nShapiro GK, et al.: A critical review of measures of childhood vaccine confidence. Curr. Opin. Immunol. 2021; 71: 34–45. PubMed Abstract | Publisher Full Text\n\nWHO: Understanding the behavioural and social drivers of vaccine uptake. WHO position paper – May 2022. Wkly Epidemiol. Rec. 2022; 97(20): 209–224.\n\nNDOH-d: National Department of Health, South Africa. COVID-19 vaccine hesitancy in South Africa: Summary of existing studies.2021 [cited 2022 14 January 2022]. Reference Source\n\nCooper S, van Rooyen H , Wiysonge CS: COVID-19 vaccine hesitancy in South Africa: how can we maximize uptake of COVID-19 vaccines? Expert Rev. Vaccines 2021; 20(8): 921–933. PubMed Abstract | Publisher Full Text\n\nWHO: World Health Organization. Data for action: achieving high uptake of COVID-19 vaccines. Interim guidance.1 April 2021. (accessed 22 December 2021). 2021.Reference Source\n\nCooper S, et al.: Vaccine hesitancy - a potential threat to the achievements of vaccination programmes in Africa. Hum. Vaccin. Immunother. 2018; 14(10): 2355–2357. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMachingaidze S, Wiysonge CS: Understanding COVID-19 vaccine hesitancy. Nat. Med. 2021; 27(8): 1338–1339.\n\nMachingaidze S, Wiysonge CS, Hussey GD: Strengthening the expanded programme on immunization in Africa: looking beyond 2015. PLoS Med. 2013; 10(3): e1001405. PubMed Abstract | Publisher Full Text | Free Full Text\n\nCooper S, et al.: Factors that influence parents' and informal caregivers' views and practices regarding routine childhood vaccination: a qualitative evidence synthesis. Cochrane Database Syst. Rev. 2021; 10(10): Cd013265. PubMed Abstract | Publisher Full Text\n\nWilson SL, Wiysonge C: Social media and vaccine hesitancy. BMJ Glob. Health 2020; 5(10): e004206. PubMed Abstract | Publisher Full Text | Free Full Text\n\nWiyeh AB, et al.: Social media and HPV vaccination: Unsolicited public comments on a Facebook post by the Western Cape Department of Health provide insights into determinants of vaccine hesitancy in South Africa. Vaccine 2019; 37(43): 6317–6323. Publisher Full Text\n\nWiysonge CS: Vaccine hesitancy in South Africa: why we need to adapt validated measures.2018. (accessed 22 December 2021).Reference Source\n\nNgcobo NJ, et al.: Human papillomavirus vaccination acceptance and hesitancy in South Africa: Research and policy agenda. S. Afr. Med. J. 2018; 109(1): 13–15. PubMed Abstract | Publisher Full Text\n\nOduwole EO, et al.: Point-of-care vaccinators' perceptions of vaccine hesitancy drivers: A qualitative study from the cape metropolitan district, South Africa. Vaccine 2021; 39(39): 5506–5512.\n\nBurnett RJ, et al.: A profile of anti-vaccination lobbying on the South African internet, 2011-2013. S. Afr. Med. J. 2015; 105(11): 922–926. PubMed Abstract | Publisher Full Text\n\nMilondzo T, et al.: Misinformation Drives Low Human Papillomavirus Vaccination Coverage in South African Girls Attending Private Schools. Front. Public Health 2021; 9: 598625. PubMed Abstract | Publisher Full Text | Free Full Text\n\nWiysonge CS, et al.: Advances in childhood immunisation in South Africa: where to now? Programme managers' views and evidence from systematic reviews. BMC Public Health 2012; 12: 578. PubMed Abstract | Publisher Full Text | Free Full Text\n\nLarson HJ, et al.: Understanding vaccine hesitancy around vaccines and vaccination from a global perspective: a systematic review of published literature, 2007-2012. Vaccine 2014; 32(19): 2150–2159. PubMed Abstract | Publisher Full Text\n\nWHO: Data for action: Achieving high uptake of COVID-19 vaccines – A guidebook for immunization programmes and implementing partners. Draft version of 26 November 2020 (Corsspondence to Ms Lisa Menning: menningl@who.int)2020.\n\nBronfenbrenner U:Ecological models of human development. International Encyclopedia of Education Oxford:Elsevier;1994; p. 1643–1647.\n\nSwann C: Action Research and the Practice of Design. Des. Issues 2002; 18(1): 49–61. Publisher Full Text\n\nGrimmer K, et al.: A South African experience in applying the Adopt-Contextualise-Adapt framework to stroke rehabilitation clinical practice guidelines. Health Res. Policy Syst. 2019; 17(1): 56. PubMed Abstract | Publisher Full Text | Free Full Text\n\nYin RK: Case study research: design and methods.5th ed.Thousand Oaks, CA:Sage Publications, Inc; 2014.\n\nNDOH-e: National Department of Health, South Africa. Update on Covid-19 (29th January 2021).2021 [cited 2021 29 January 2021].Reference Source\n\nFaul F, et al.: Power 3: A flexible statistical power analysis program for the social, behavioral, and biomedical sciences. Behav. Res. Methods 2007; 39: 175–191. PubMed Abstract | Publisher Full Text\n\nGroenewald C, et al.; A qualitative report on learners’ experiences and perceptions of the ‘It starts today’ intervention.2018.Reference Source\n\nGroenewald C, Essack Z: Bring a picture, song or poem: the expression session approach. Paper presented at the 32nd International Congress of Psychology (ICP), Prague, Czech Republic (virtual attendance), 18-23 July 2021, in 32nd International Congress of Psychology (ICP). Prague, Czech Republic. 2021.\n\nPeabody CG: Using Photovoice as a Tool to Engage Social Work Students in Social Justice. J. Teach. Soc. Work. 2013; 33(3): 251–265. Publisher Full Text\n\nBraun V, Clarke V: Using thematic analysis in psychology. Qual. Res. Psychol. 2006; 3(2): 77–101. Publisher Full Text\n\nBazeley P, Jackson K: Qualitative Data Analysis with NVivo. 2nd ed.London, UK:SAGE Publications Ltd.;2013.\n\nCharmaz K: Constructing Grounded Theory: A Practical Guide through Qualitative Analysis. 2nd ed.London, UK:SAGE Publications Ltd.;2006.\n\nKoen J, et al.: Payment of trial participants can be ethically sound: moving past a flat rate. S. Afr. Med. J. 2008; 98(12): 926–929. PubMed Abstract\n\nNHREC: Payment of trial participants in South Africa: Ethical considerations for Research Ethics Committees South Africa:National Health Research Ethics Council;2012.\n\nWiysonge CS, et al.: COVID-19 vaccine acceptance and hesitancy among healthcare workers in South Africa. Expert Rev. Vaccines 2022; 21: 549–559. Publisher Full Text\n\nKatoto P, et al.: Predictors of COVID-19 Vaccine Hesitancy in South African Local Communities: The VaxScenes Study. Vaccines (Basel) 2022; 10(3). Publisher Full Text\n\nWiysonge CS, et al.: Community-based Case Studies of Vaccine Hesitancy and the COVID-19 Response in South Africa; The VaxScenes Study. medRxiv 2022; p. 2022.02.21.22271272.\n\nWiysonge CS, et al.: Questionnaires for the “Community-based case studies of vaccine hesitancy and the COVID-19 response in South Africa”. Figshare 2022: Publisher Full Text"
}
|
[
{
"id": "158387",
"date": "19 Jan 2023",
"name": "Kerrie Wiley",
"expertise": [
"Reviewer Expertise Understanding immunisation behaviour and it's implications for policy and practice."
],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 protocol. The manuscript is well-written, and the described study protocol is of high interest and importance. Overall, I would suggest a close review of the terminology used throughout the protocol, and be very clear on the difference between vaccine uptake (behaviour) and vaccine hesitancy (a motivational state that is one of many drivers of vaccine acceptance or rejection). Throughout the manuscript there appears to be a focus on understanding vaccine hesitancy, however the chosen conceptual framework and methods (the Social Ecological model, and the BeSD survey) focus on understanding the many contextual drivers of vaccine uptake behaviour which includes but is not limited to vaccine hesitancy, suggesting to me that uptake rather than hesitancy might be focus of the study. Please find my section-specific comments below.\nABSTRACT: The abstract is well-structured, and adequately summarises the paper.\nINTRODUCTION: The introduction summarises the context-specific situation in South Africa regarding COVID-19 response, including the need for adequate vaccine uptake, going on to identify vaccine hesitancy as a main challenge for a successful COVID-19 vaccine programme in South Africa, and uses the WHO BeSD Working Group definition of vaccine hesitancy.\n\nI think it’s important to be very clear about the difference between hesitancy and uptake here. I agree with the statements that there are many elements that could contribute to vaccine hesitancy, and that issues like people’s previous experiences will contribute hesitancy and uptake. While I agree that the impact of context-specific factors affecting the “enabling environment” will be comparatively greater in SA, I would be cautious in asserting that the impact will be on hesitancy. My reasoning is, here we explicitly use the definition of hesitancy as a motivational state. Under this definition, hesitancy is not a behaviour, with the behaviour of interest being vaccine uptake (i.e. uptake, rather than intention or willingness). If we apply the WHO Behavioural and Social Drivers of Vaccination framework, the “enabling environment” factors such as the given example of transport cost/availability would sit under the “Practical Issues” domain, which is separate to the motivation domain (hesitancy), but combined with hesitancy and other considerations will contribute to the behaviour of vaccine uptake. Practically speaking, there are hesitant people who still go ahead and take up vaccines, and there are other people who are not hesitant at all and remain unvaccinated due to things like those practical issues / the enabling environment that are mentioned.\nMETHODS: The methods are very well-described.\n\nConceptual framework: the SEM is very appropriate as a conceptual framework. Here again, I encourage the authors to be careful in separating hesitancy (motivation) and vaccine uptake (behaviour). This paragraph discusses the different interventions needed to implement behaviour change (i.e. increase vaccine uptake), but then the description of Box 1 describing how the SEM can be applied to addressing vaccine hesitancy. If behaviour (vaccine uptake) is the outcome of interest in this study (and the role hesitancy might play in that among other things), I would consider replacing “hesitancy” with “behaviour change” in labelling and discussing this.\nStudy design: Case study methodology is appropriate for this study. Again, I question whether this is about exploring the “how and why” of possible vaccine hesitancy, or exploring the how and why of vaccine uptake, and the possible role of vaccine hesitancy, among other drivers. Study phases: Here again we see a description of “deeper investigation of vaccine hesitancy in phase 2”, however Phase 2 uses the BeSD survey, which was specifically designed to measure a range of drivers of vaccine uptake (behaviour), and doesn’t focus solely on hesitancy.\nData Analysis: Regarding the sentence “Logistic regression models will also be used to evaluate the association between selected characteristics and vaccine hesitancy as well as adherence to NPIs”. The BeSD survey in it’s published form does not cover NPIs. Was the survey modified to include items that measure NPI adherence and related drivers of that behaviour? If so, how were the items selected or developed and were they validated? Also, is vaccine hesitancy (i.e. the “motivation” item on the BeSD survey) the intended outcome variable for the regression? Or will it be COVID vaccine uptake?\nDISCUSSION AND CONCLUSION: As with the preceding sections, I found myself asking, is the focus vaccine hesitancy? Or vaccine uptake and it’s drivers, including hesitancy? The BeSD tools are designed to measure the latter. Since the study was conceived in 2021 the BeSD tools have been validated in some African nations (see https://apps.who.int/iris/bitstream/handle/10665/354460/WER9720-209-224-eng-fre.pdf?sequence=1 ), but as far as I’m aware not in South Africa. This section could be updated to reflect this.\nThis is an impressive mixed methods study that I think will yield meaningful context-specific results for the communities that are involved. All that is needed is clarification about whether hesitancy is truly the focus, or whether it’s the relative role of hesitancy in vaccine uptake decisions.\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? Not applicable",
"responses": []
},
{
"id": "190253",
"date": "09 Oct 2023",
"name": "Srikanth Umakanthan",
"expertise": [
"Reviewer Expertise COVID19 infections",
"vaccine hesitancy",
"molecular genetics."
],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe manuscript is well written and provides a good insight into the role of vaccine hesitancy, proving to be the major hindrance against successful vaccination.\nAbstract:\nThe abstract provides a brief outline on the study and its findings. However, specific details about methodology, sample size, data collection process and potential bias needs to be mentioned.\n\nIntroduction and discussion:\nThe introduction should include a global perspective and then move towards the regional impact of COVID-19 (refer and cite — Umakanthan et al., 20201).\n\nCompare the study with other recent relevant studies to showcase the similarities and differences.\n\nA more specific critical analysis should be provided along with data interpretations in the results section. Mention and address the role of bias, recommendations, and limitations in a broader extent.\n\nThe data validation tool or the pilot model should be explained.\n\nIndicate studies done in South Africa or in Africa for better understanding.\n\nIndicate the knowledge gap the authors aim to bridge with this study and those differing from the current literature.\n\nResults:\nInclude more of figures and tables for better understanding and avoid replication of data in the results text.\n\nRecommendations, strengths and limitations to be included as a separate section.\n\nEthical statements can be added along with methodology section.\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? Yes",
"responses": []
}
] | 1
|
https://f1000research.com/articles/11-1514
|
https://f1000research.com/articles/11-1509/v1
|
13 Dec 22
|
{
"type": "Policy Brief",
"title": "Areas of research interest: joining the dots between government and research at last?",
"authors": [
"Kathryn Oliver",
"Annette Boaz",
"Giulia Cuccato",
"Annette Boaz",
"Giulia Cuccato"
],
"abstract": "Background: With the aim of making it easier for researchers to produce policy-relevant research, the UK Government now requires all departments and arms-length bodies to publish annually-updated statements of their evidence needs, called ‘Areas of Research Interest’ (ARIs). We describe how ARIs are produced, and how they are used to support this aim.\nAims and objectives: In this paper we offer a description of ARIs and their development by UK governmental departments, and an assessment of how different stakeholders, including academia and funders, have responded to or otherwise used the ARIs.\nKey conclusions: ARIs are a mechanism for organisations to share their research interests with external audiences in the form of a published document. In addition to this primary aim, they also have a much broader set of uses, including connecting departments with each other and helping intermediaries shape engagement plans. All groups would benefit from more robust evidence to choose effective engagement mechanisms, and more can be done to make the ARIs discoverable and useable. Overall, the ARIs are a useful tool to illuminate, and begin to connect different parts of the research-policy system.",
"keywords": [
"evidence use",
"Areas of Research Interest",
"policy"
],
"content": "Introduction: what are areas of research interest?\n\nAlthough governments, funders and researchers often share a desire for more evidence to support social change, there are significant organisational and procedural barriers in the way. One often-mentioned barrier to evidence use in policy is a lack of policy relevant research (Armstrong et al., 2006; Oliver et al., 2014; Cairney, 2016; Wye et al., 2019). One way of addressing this barrier is to make it easier for researchers to know about policymakers’ research priorities. In the UK, government departments identify priority research gaps called Areas of Research Interest (ARIs). The Nurse Review into the UK Research Councils (Nurse, 2015) identified a strategic need across government departments to “maintain[] ‘statements of need’, in terms of the most important research questions confronting the Departments” (p.3, Nurse, 2015). By enabling other government departments, funders, and researchers, ARIs were intended to enable policymakers to access relevant evidence and expertise more easily. A similar initiative has been proposed in the US, called the Government Learning Agendas1, where all departments and arms-length bodies are required by the Evidence Act (2019) to publish their knowledge needs.\n\nMany policy and practice organisations including government departments of course communicated their evidence needs before these initiatives (e.g. in Defra’s evidence plan from 2006). However, the ARIs are the first time that every department has been required to publish these publicly, update them annually, and invest resources into their production and use; in effect, 'systematising and embedding the articulation of research interests as part of the fabric of policy making.\n\n\nThe role of our team\n\nIn the UK, the Government Office for Science (GOS) supports the Government Chief Scientific Adviser (GCSA), and holds a cross-government remit to support science capability across departments. It relies on 'soft power' networking and influencing rather than mandating. It also holds the policy for Areas of Research Interest. As part of this remit, Giulia Cuccato led a team of civil servants in the Government Office for Science (GOS) to develop guidance for departments in developing their ARIs, and supporting and tracking their ARI-related activities. Alongside this work, Kathryn Oliver and Annette Boaz have been embedded in GOS to explore and support better production of ARIs, and more effective engagement with them by funders, researchers and intermediaries. We have been involved in supporting the development of ARIs across the UK government, in helping departments use ARIs to access relevant evidence and expertise, and in researching how ARIs could be optimised to support the research-policy system.\n\nOur reflections here are based on our work since 2018 (GC) and 2019 (KO and AB) which has involved working closely with analyst and policy teams in departments across government, and with intermediaries such as the University Policy Engagement Network (UPEN), the National Academies, the What Works Network, and individual universities and researchers, in a practical knowledge mobilisation programme. We draw on these experiences to discuss the strengths and weaknesses of ARIs, and our recommendations to improve their effectiveness as a systems-level intervention.\n\n\nPolicy outcomes and implications\n\nThe ARIs have great potential to enable funders, government, research organisations, researchers, and intermediaries to work together in a more effective way. We have observed that merely producing ARIs is not a sufficient intervention; instead, it requires skilled mobilisation work by people within all these organisations to be able to optimise their production and use. Below, we describe how ARIs are produced, what ARI-related engagement occurs, and how they function as a systems level intervention.\n\nWhen GCSA Patrick Vallance joined GOS in 2018, one of his main goals was to review and increase the scientific capability within government (Government Office for Science, 2019). This review published recommendations, one of which set out the requirements for annually-updated ARIs to be published by each department. There was considerable variation between government departments. Some departments had existing strong science systems and plans which included an articulation of their research needs, and others had never attempted to map or describe their internal science systems before.\n\nThe team with GOS (GC, plus a full-time intern and resources from HEO and SEO level staff) worked with departments to identify the maturity of their science systems, establish their history of developing science plans or equivalents, the connectedness of their science and analysis teams with the policy function within each department, and their consequent ARI support needs. The ARI Fellows (KO and AB) worked with departments offering tailored advice about how to produce and publish ARIs, and how to engage effectively with academics during and after the production process.\n\nAs of summer 2022, all departments and arms-length bodies have published at least one version of their ARIs, with at least ten updated in the last two years. Over 1500 ARIs have been published which vary greatly in terms of content, production and associated activities (Haddon and Sasse, 2018).\n\nFor most departments, the production of ARIs was a primarily internal process. For example, the Departmental Chief Scientist might task the science and analysis team within a department with drafting a set of ARIs. Some departments connected the development of ARIs with their departmental science strategy which set out projected R&D spending for the financial year, and with policy priorities for the department. Some departments engaged with external experts to frame, prioritise and shape their ARIs, for example through their Science Advisory Councils. Others took a highly proactive approach. For instance, Defra held a two-day conference in 2018 with representatives from industry, research and practice to develop their initial ARIs. Once drafted, ARIs are signed off by the relevant directors, the minister, and then published on the UK government website: gov.uk2.\n\nARIs were intended to identify strategic research priorities for departments, but in practice we have found that they have a much broader set of uses. We found that departments use them to improve internal working and relationships, to implement the agenda of the Chief Scientist, to support other governmental processes such as spending reviews, the Integrated Review, and the Science Capability review. For some they are a reflection of their policy priorities; for others an articulation of the activities and structures of their internal science system; a statement of likely research commissioning priorities; and/or a statement of research areas around which they would welcome collaboration or input. However, the end products appeared to be mostly appropriate for the departments in question. By and large, they were seen as useful internal tools to negotiate and communicate with policy colleagues around budgets and priorities, and useful external tools to solicit help.\n\nUniversities and academics find them useful to plan engagement activities such as workshops and fellowships, but often tend to view them as poorly-written research questions. ARIs can help the research community to understand what government departments want from them. This happens most effectively when there are opportunities for dialogue or a clear narrative about the policy history behind each ARI.\n\nIdentifying relevant expertise and research is a real challenge for government departments, particularly where resources are limited. Framing problems is an important step for departments, because it dictates what research and which experts are considered relevant and appropriate. We found that officials in government departments were committed to addressing the challenge of diversity and inclusion in academic-policy engagement, but we unsure how best to go about improving practice in this area. Departments took different approaches to identifying relevant experts and evidence, ranging from using existing formal structures such as Scientific Advisory Committees and Chief Scientific Advisors, to effectively outsourcing the selection of candidate experts to partners such as the UK Universities Policy Engagement Network. We also found that universities and researchers often reached out directly to departmental officials. While well-intentioned, these approaches often created very significant amount of engagement work for officials on top of their usual engagement activities such as advisory councils. We observed that this had the potential to create competition, duplication and wasted resource on all sides, which is consistent with the literature on academic-policy engagement (Oliver et al., 2022).\n\nDepartments had no clear process to identify which engagement mechanism was the most appropriate for each ARI or set of ARIs. Using a survey of the officials most frequently engaged with ARI-related work, we found over 22 engagement mechanisms were used by departments (see Table 1). Most departments tend to rely on tried and tested approaches (such as commissioning research projects).\n\nWe observed that there was scope to broaden the menu of options available to departments when planning and implementing their ARI-related engagement. For example, officials reported primarily using ARIs to commission new research projects or support the generation of new research in other ways. However, we found that there is often – even usually - significant research and expertise already available. In these cases, evidence syntheses or interactive roundtables might be more appropriate and useful methods for gathering relevant research. However, planning a detailed stakeholder engagement plan around ARIs is skilled work which takes resource to plan and execute. For example, skilled staff are needed to support external experts to operate effectively within a policy environment; external experts often require training; time and effort from all side is required to put together a Workplan acceptable to all; and finally, there are operational barriers around political sensitivity, contracts, and finances which take time to resolve.\n\nARIs work well as an external articulation of research and evidence needs for departments. They offer funders, intermediaries and researchers’ insights into what departmental research agendas. Universities and intermediaries in particular have used ARIs to develop their own strategic engagement plans (see, e.g. Heckels, 2020). Most departmental ARI documents now contain contact details as well as ‘asks’ and ‘offers’ for each ARI. This makes it easier for funders, intermediaries and researchers to know how to respond (e.g. by getting in touch for a conversation, arranging a research collaboration or responding to a research tender).\n\nARIs as a systems intervention: The ARIs were proposed to encourage the production of more policy-relevant research. This has been described as a ‘deficit model’, suggesting that if decision-makers had better evidence, their decisions would improve. This model has been widely criticised as being based on some fundamentally flawed assumptions about how decision-making works (Jones and Crow, 2017) and on how evidence informs that process (Locke, 2002). The ARIs may have been planned to address this illusory ‘deficit’, but in practice perform a far greater range of functions which help to connect the policy research system in complex ways. We have seen the impact of ARIs in:\n\n• Improved understanding of what resources are required, and where, to optimally support ARI work and R&D across government\n\n• Connecting academics with government officials, e.g. Welsh Centre for Public Policy with colleagues in DHSC and DWP; What Works Centre for Cities connecting with government officials across departments\n\n• Influencing new Strategic Priority Fund bids (e.g. NERC SPF programme shaped by the Land Use group to broaden beyond biodiversity to include more social and urban research)\n\n• Enabling discussion between funders and government, e.g. STFC with GOS work on security and resilient; DWP with ESRC grant managers; Innovate UK with GOS colleagues to work on cross-government and UKRI responses to the spending review\n\n• Influencing future funding programmes which follow the themes of our knowledge mobilisation events (e.g. Just transition/vulnerable communities by BA) or drew on our knowledge of ARI mobilisation to inform design (ESRC Policy Fellowship scheme)\n\nThe true value of ARIs may be in illuminating the ways in which the research-policy system is connected, and how we can intervene most effectively to support this system.\n\n\nWeaknesses of the ARIs: systems challenges\n\nNot everything can be or is articulated as an ARI: ARIs are not able to articulate the totality of departmental research needs. For some departments, this is due to political or operational sensitivity, and for others, they prefer to only publish ARIs on topics where they are currently seeking external input. It would be a mistake, therefore, to think of ARIs as a complete and exhaustive list of the topics on which government is seeking input.\n\nARIs are not research questions: Academics frequently describe ARIs as poorly written research questions. An alternative, more useful phrase might be “research needs”, which helps to give the impression that there is a process attached to them, that they are valued, and broader than research questions. They are more usefully thought of as topics for conversation.\n\nARIs are difficult to access and analyse: By 2018, most departments had published at least one version of their ARIs, which then sat on the government website in pdf or html formats. There is as yet no easy way to search for ARIs by topic, department or year, which makes it difficult for researchers to identify relevant topics or potential collaborators This also means that departments are not easily able to identify shared cross departmental areas of interest.\n\nFinding relevant evidence and expertise takes time and work: While some departments had resources dedicated to engagement around the ARIs, others did not. While relevant research often exists (as bodies of primary research, in research and practice communities, or in ongoing funding investments), this knowledge is often inaccessible, being behind paywalls or requiring time and skill to find and absorb.\n\n\nActionable recommendations\n\nAreas of Research Interest are a helpful intervention, as a cog in the system which may help us make and use evidence more effectively to inform policy. They are a positive development, without being a silver bullet solution to the knotty problem of evidence production and use. However, much could be done to optimise their production and use.\n\nFunders need to support knowledge exchange and evidence synthesis as well as primary research relevant to these areas. At present, funders are often not able to easily access their portfolios of existing investments, which means it is difficult for them to help departments access ARI-relevant existing research or relevant experts. Funders do not appear to be able to track use of ARIs in proposals, and it is not clear how they shape future investments. Finally, funders do not appear to being using ARIs as a basis to identify shared interests) and where they do fund knowledge exchange activities, they do so in an uncoordinated and non-strategic way. Hardly any funders invest in research about evidence production and use, which would produce empirical evidence to guide these activities.\n\nIntermediaries: Intermediaries (such as the National Academies, selected What Works Centres such as the Wales Centre for Public Policy) often do have both skilled and resourced individuals who work across academic-policy boundaries. Further investment in brokers and intermediaries would be an effective way to strengthen research-policy engagement. Where there is significant staff turnover in government (as civil servants move roles) Intermediaries can become the repository for organisational memory and could advise departments on their own policy and research engagement history. However, this work is often essentially unpaid, and there is no easy way for the skills and careers of knowledge mobilisers in these organisations to be nurtured. Intermediaries occupy a really important place in the academic policy landscape, enabling them to convene and facilitate cross-field and interdisciplinary discussions. We found that Intermediaries were really valued, able to access experts, bring in diverse voices and knowledge types. However, the brokering organisations we saw (National Academies, What Works Networks, UPEN) did not represent all academics and communities.\n\nResearchers and universities need to incentivise effective policy engagement. individual researchers often lack structures to help them understand policy need, so despite the existence of the ARIs, they struggle to address policy agendas. They are not well-trained or incentivised to engage well, and where they do, they tend to actively push experts and expertise out via marketing and PR approaches, rather than thinking about the overall science system. This leads to competition between them for officials’ time, which is inefficient and drives inequity and a lack of diversity in the science advisory system. Training is often about increasing volume and impact of individual researchers or projects. We observed that almost all the university-led projects and engagement activities sought to push university agendas (e.g. institutional profile) or researcher agendas (e.g. individual careers, or importance of particular pieces of evidence). This is not helpful for policy colleagues, who then have to analyse where engagement is most useful and least costly. There is also a growing infrastructure to support knowledge mobilisation within universities, and interestingly across them too. The University Policy Engagement Network is an opportunity to collaborate on and share knowledge exchange training, best practice, and a chance to offer government a more diverse set of experts to draw on.\n\nGovernment departments: need to be clear what is being asked and offered for each ARI. Their current resources and histories of engagement shape their ARI planning, but a broader range of engagement mechanisms would make academic-policy engagement more efficient and effective. Departments may benefit from more support to develop and implement engagement plans effectively, minimising duplication and maximising value for money. There is also a need to track the use of ARIs to understand how they are used and demonstrate to departmental colleagues the value of investing time in ARI development and academic engagement.\n\nAll groups would benefit from a more robust evidence base about how to engage most effectively There is considerable scope to improve the process of selecting appropriate next steps, and to expand the range of research-policy engagement mechanisms used. In general, seeking to complement existing structures and processes is most efficient and leads to least disruption.\n\nMaking ARs discoverable: in their current form (as published ARIs documents from each government department) they can be hard to search and access (especially for cross departmental issues). Making the ARIs accessible would enable researchers and funders to identify trends, themes and topics to inform their future and present work. Government officials would be able to discover areas of shared interest with other departmental colleagues and work on shared priorities together. This would be a great public resource.\n\nUnderstanding the impact of ARIs: The impacts we identified from our ARI-related knowledge mobilisation are not a comprehensive overview of how they are shaping research, engagement and investment activities. It would be useful to have a more exhaustive exploration of who is using ARIs, how, and for what purpose. As we note above, it is unlikely that ARIs will ever be able to capture all governmental research needs or engagement activities. For example, they may be more useful at capturing topics which could inform policy over the medium to long-term than for short term problems.\n\nShared problem-framing and priority-setting: ARIs offer an opportunity for government to think and articulate its strategic aims. To operationalise these aims takes organisational capability which needs to be built over time, both within government and in its external partners. This might be achieved by a cross-government initiative or department taking responsibility for identifying areas of shared interest, a process for prioritisation, and/or a place to optimise engagement between academia and government.\n\n\nConclusions\n\n\n\n• By stating their evidence needs, government departments make it easier for researchers to produce or provide relevant research.\n\n• ARIs perform a range of useful functions helping to connect parts of the policy research system in different ways.\n\n• Improving the accessibility of ARIs through a searchable database would increase their utility to all users.\n\n• Government, researchers, funders and intermediaries would benefit from a more robust evidence base about how to engage most effectively to optimise the use of ARIs.\n\nThe ARIs are a useful step forward to a more research-informed policy culture. We have highlighted some of the work which is needed to make ARIs useful, and identified points within the system where intervention and support would be beneficial. To make this happen, investment and resource will be required. Overall, these ARIs are an interesting model for other governments wanting to improve research production for and use in policy.\n\n\nAuthor contribution\n\nAll authors developed the ideas for the paper and approved the final manuscript.",
"appendix": "Data and software availability\n\nAll the ARIS are available on https://www.gov.uk/government/collections/areas-of-research-interest . No primary research data was analysed to produce this paper.\n\n\nAcknowledgments\n\nWe wish to thank the other members of our team within GOS, all the participants in our knowledge exchange activities from across government, funders, intermediaries, and academia, and our team within the ESRC who have supported this activity. All of you have helped us to develop our understanding of ARIs and their role across government and beyond.\n\n\nReferences\n\nArmstrong R, et al.: The role and theoretical evolution of knowledge translation and exchange in public health. J. Public Health. 2006; 28: 384–389. PubMed Abstract | Publisher Full Text\n\nCairney P:Conclusion: How to Respond to the Limits of EBPM. The Politics of Evidence-Based Policy Making. London:Palgrave Macmillan UK;2016; pp. 119–133. Publisher Full Text\n\nGovernment Office for Science: Realising our ambition through science: a review of Government Science Capability.2019.Reference Source\n\nHaddon C, Sasse T: How government can work with academia. London:2018.\n\nHeckels N: Engaging with UK Government Areas of Research Interest: learning and insights from the Universities Policy Engagement Network.2020.\n\nJones M, Crow D: How can we use the “science of stories” to produce persuasive scientific stories. Palgrave Commun. 2017; 3(1): 53. Publisher Full Text\n\nLocke S:The public understanding of science - A rhetorical invention. Science Technology and Human Values. Thousand Oaks, CA:Sage PublicationsSage CA;2002; 87–111. Publisher Full Text\n\nOliver K, et al.: A systematic review of barriers to and facilitators of the use of evidence by policymakers. BMC Health Serv. Res. 2014; 14(1): 2. PubMed Abstract | Publisher Full Text\n\nOliver KA, et al.: What Works in Academic-Policy Engagement? Evid. Policy. 2022; 18(4): 691–713. Publisher Full Text\n\nWye L, et al.:Knowledge brokers or relationship brokers? The role of an embedded knowledge mobilisation team. Evid. Policy. Univ Bristol, 1-9 Old Park Hill, Bristol BS2 8BB, England:Policy Press;2019; 15(2): 278–292. Publisher Full Text\n\n\nFootnotes\n\n1 www.evaluation.gov/evidence-plans/learning-agenda/\n\n2 https://www.gov.uk/government/collections/areas-of-research-interest"
}
|
[
{
"id": "158048",
"date": "06 Feb 2023",
"name": "Hannah Durrant",
"expertise": [
"Reviewer Expertise Evidence use in policymaking",
"knowledge mobilisation",
"policy design and evaluation",
"loneliness and community wellbeing."
],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 valuable and well-written paper. It provides a succinct and much needed assessment of where government areas of research interest (ARIs) have come from in the UK, how they are being produced and used, and their strengths and weaknesses. It does this from a unique combination of insider-researcher perspectives and, in doing so, can claim to practise what it preaches in terms of active and engaged forms of knowledge mobilisation (indeed I think more can be made of this). Actionable recommendations are provided for all stakeholders – many stress the importance of intermediary mechanisms for knowledge mobilisation to bring government evidence needs together with experts and expertise to understand what evidence exists and what new knowledge could be produced to inform decision-making. They raise the urgent need for more robust evidence on what mechanisms for knowledge mobilisation are most effective, when, under what conditions, and why.\nI have a few comments and suggestions for areas to strengthen the paper further, which are outlined below. There are also a few, small typographical points which may be picked up in a robust copy edit, e.g., some words missing from the sentence in the first paragraph that starts “By enabling other government departments, funders, and researchers…”; there is a quotation mark in front of the word “systematising..” in paragraph 2; at the bottom of page 5, it should be ‘Wales Centre…’ not \"Welsh Centre…\"; top of page 6, \"BA\" should be 'AB'; middle of page 6, it should be “funders do not appear to be using…” rather than “being using…”; etc. These are small and infrequent but would perfect the paper.\nComments and suggestions The paper would benefit from elaborating a bit more (e.g., within the section on the role of the team) on the ARI fellowship design, and the specific activities undertaken that generated the insight that underpins the claims made later in the paper (e.g., in the middle of page 4 about what universities and academics find useful about ARIs, how UPEN functions as an intermediary, and points about competition and duplication arising from direct engagement).\nIt would be worth making it clear for a broad base of readers that two of the authors (KO and AB) were/are embedded researchers.\n\nIt would also be very valuable to provide a brief explanation of embedded research and any assessment of evaluation as one among many mechanisms to enhance knowledge mobilisation. Given that it is a highly relational and engaged approach to brokering knowledge to support decision-making, it seems a highly appropriate approach to choose, particularly in light of limitations raised about efforts to merely ‘push’ research evidence out to policy. That said, the approach is not without difficulties (some of which may be captured in points made about the transactional costs of engagement and the lack of support and recognition for these roles and approaches). Is there an opportunity to reflect on this approach in the context of discussion about the benefits and challenges of mechanisms for knowledge mobilisation in response to government ARIs? The Wye et al. (2019) paper already cited could provide a starting point for conceptualising embedded research/ers. More importantly, it would be valuable to outline how the team designed their approach to maximise the potential of their fellowships to achieve the aims and avoid any pitfalls.\n\nI think it would be useful to provide a little more detail on the nature of the work of the team with government department analysts and policy teams, as well as with intermediaries, universities and researchers, in order to underpin the perspectives on ARIs that you report in the section on what ARIs are for. What kinds of interactions happened? How often? What was the purpose? Were there any limitations that should be taken into account?\nYou note variation between government departments in the production of ARIs, differences in what they are intended for, and that not all government ‘evidence needs’ are/can be articulated as ARIs. These observations suggest implications for whether ARIs actually signal evidence need on the most important departmental research questions, as intended in the Nurse Review (N.B. could you provide the reference for the Nurse Review, 2015?). Even if we accept a flawed ‘deficit model’ that misrepresents policymaking processes (your critique here is very valid), it remains unlikely that evidence can inform decisions if the most important evidence needs are not articulated. It may be worth reflecting on this, alongside other points made about whether ARIs are likely to generate/stimulate the mobilisation of the most relevant and useful evidence for addressing policy priorities and to meet the stated ultimate aspiration for evidence to inform social change. This may strengthen the point that ARIs should be seen as providing a starting point for focused conversation and that their impact (and limits) under these conditions need to be better understood in order to inform a multi-method approach (including methods more likely to get to the most important policy research questions) for strengthening research-policy engagement on important topics of government priority that ARIs don’t cover.\nIn the section on 'Strengths of ARIs: ARIs as a systems intervention', I wondered if the team had observed any impacts of ARIs in terms of enabling university-based intermediaries (e.g., policy engagement support within individual universities or within UPEN) to focus the ways they seek to coordinate and connect academics to government officials? I do not think the Wales Centre for Public Policy (WCPP) provides a good example of how ARIs work to connect academics with government officials. This is not core to WCPP's work or a typical activity for the Centre, but good examples are useful and may be found elsewhere.\nFinally, given that the paper stresses the importance of intermediaries and knowledge mobilisers/brokers (albeit underpinned by an urgently needed evidence base on what works), I think the conclusions would be strengthened by adding a point on actively supporting knowledge mobilisation around ARIs, which requires action and investment by funders, universities, intermediaries and government. The buy-in of these stakeholders may be crucial for plugging the evidence gap on effective mechanisms for better research-policy engagement.\nI think these comments are minor and overall this paper makes an important contribution to better understanding engagement with government ARIs. Thank you again for an enjoyable read.\n\nDoes the paper provide a comprehensive overview of the policy and the context of its implementation in a way which is accessible to a general reader? Yes\n\nIs the discussion on the implications clearly and accurately presented and does it cite the current literature? Partly\n\nAre the recommendations made clear, balanced, and justified on the basis of the presented arguments? Yes",
"responses": []
},
{
"id": "176614",
"date": "16 Jun 2023",
"name": "Sarah Ball",
"expertise": [
"Reviewer Expertise Public policy and knowledge translation practices"
],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 piece, which definitely presents a comprehensive overview of the policy and the context of its implementation in a way which is accessible to a general reader. The aim is descriptive, and it does an excellent job of achieving that aim. The development of ARI's, and the barriers and potential facilitators to their use, is an area which will be of great interest to researchers and practitioners in the UK, but also for other countries looking to facilitate collaboration between government and the research sector. The findings are targeted and actionable, with valuable learnings for funders, intermediaries, governments and researchers. These findings are clear, balanced and well supported by the descriptive research that precedes it.\nThe need for more research is also very clear. Given this, I would have appreciated some indication of whether this was part of a larger project which has produced or will be producing research papers/journal articles which step out methodology more clearly. The findings are useful, but I would love to see some more detailed discussion and analysis (while recognising this is not the aim of this paper). Some claims such as 'academics frequently describe ARI's as poorly written research questions' would be worth providing more evidence for, or making more equivocal, in light of this. Without any methodology section, I have some reservations about making claims like that otherwise.\n\nMy only other note is that there are a few typos in this proof:\np4 para 5 'we unsure', should be 'were unsure'\n\np6 para 4 'collaborators This' missing full stop.\n\np6 para 8 ' ) Intermediaries' missing full stop.\n\np6 para 9 'engagement. individual' Individual should be capitalised.\n\np7 para 4 'AR's' should (presumably) be ARI's.\n\nDoes the paper provide a comprehensive overview of the policy and the context of its implementation in a way which is accessible to a general reader? Yes\n\nIs the discussion on the implications clearly and accurately presented and does it cite the current literature? Yes\n\nAre the recommendations made clear, balanced, and justified on the basis of the presented arguments? Yes",
"responses": []
}
] | 1
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https://f1000research.com/articles/11-1509
|
https://f1000research.com/articles/11-134/v1
|
02 Feb 22
|
{
"type": "Study Protocol",
"title": "The effectiveness of a sexual assault nurse examiner-grounding program (SANE-GP) on knowledge, skill and practice regarding sexual assault examination (SAE) among nurses working in a tertiary care hospital in Udupi district, India: A study protocol",
"authors": [
"Renjulal Yesodharan",
"Vinod Nayak",
"Tessy Jose",
"Vikram Palimar",
"Anice George",
"Renjulal Yesodharan",
"Tessy Jose",
"Vikram Palimar",
"Anice George"
],
"abstract": "The medico-legal care of victims of sexual assault is very challenging, and requires specific knowledge and skills. Professionals in the emergency departments of hospitals might not have specialised training in forensic science. Nurses have a very significant role in these settings, but they lack any formal forensic training. This study aims to develop a sexual assault nurse examiner-grounding program (SANE-GP) for Indian nurses to inculcate knowledge and skill regarding sexual assault examination. The study adopts a three-stage Delphi technique to develop the training module and uses a time-series design to evaluate the effectiveness of the program. A questionnaire on nurses’ knowledge on sexual assault examination (KQSANE-I) will be developed in phase-I and subsequently used in phase-II. The protocol of SANE-GP will help the medical community to implement the program across India. The implementation of SANE-GP can also help to start a sexual assault nurse examiner network.",
"keywords": [
"Forensic nursing",
"Forensic Science",
"SANE",
"sexual assault",
"sexual violence"
],
"content": "List of abbreviations\n\nASO: Alternative Sexual Orientation\n\nCrPC: Criminal Procedure Code\n\nDAC: Dissertation Advisory Committee\n\nKQSANE-I: Questionnaire on nurse’ knowledge about sexual assault examination\n\nPOCSO: The Protection of Children from Sexual Offences Act\n\nSAE: Sexual Assault Examination\n\nSAFE: Sexual Assault Forensic Examination\n\nSANE: Sexual Assault Nurse Examiner\n\nSANE-GP: Sexual Assault Nurse Examiner-Grounding Program\n\nWHO: World Health Organization\n\n\nIntroduction\n\nSexual violence is a huge concern and has malignantly affected all strata of our community regardless of age, gender, race, ability, and social status. However, the estimates of incidents of sexual violence around the globe suggest that there is a predominance of sexual violence against women in comparison to the sexual violence against men. The annual report “Crime in India – 2019” by the National Crime Record Bureau (NCRB) indicated that a total of 4,05,861 cases of crime against women were registered in India during 2019, which includes 7.9% of rape cases. The crime rate registered per lakh women population was 62.4 in 2019 in comparison with 58.8 in 2018 (National Crime Records Bureau (NCRB), 2019). Sexual violence affects many aspects of the victim’s life including personal safety, their family, interpersonal relationships, finance, and work environment, and makes the victim undergo perplexity of investigation and legal trial. Unfortunately, in some cases the medical, legal and law enforcing agencies operating in the country and the society itself, make the victim feel the sexual assault was their fault and re-victimise them for someone else’s cruelty. (Relyea and Ullman, 2017). Most of the time, the trauma they are already undergoing, is much less than the trauma they receive from the system — a system that is supposed to help them (Maier, 2008). Sexual offenses are under-reported to law enforcement agencies due to fear of retaliation and humiliation (Shanmugam, 2013; Flecha, 2021). A comparative study of the NCRB data on rape and the National Family Health Survey shows that only 0.6% of women who faced sexual violence filed a complaint to the police (Rukmini, 2014). The legal system of India is common to all states and union territories. The judiciary is independent and not governed by the federal government. The time taken for a case to come to trial in the Indian court is getting longer. Crimes against women and an increase in the incidents of criminal activities have contributed to the workload of the judiciary system (Jaithlia and Maheshwari, 2020).\n\nWhen victims are being re-victimised by the system, most of them will not go to the hospital and report that they were sexually assaulted and are also not willing to follow the criminal justice system (Maier, 2012). This causes a significant underestimation of the prevalence of crimes against women and children (Murshid and Bowen, 2018; Jennings, Powers and Perez, 2021). The Criminal Law Amendment Act 2013 widened the definition of rape and acknowledges the right to get medical and forensic attention to all victims and survivors of sexual assault by health care centres in public as well as private sectors. Failure to provide health care services is now considered an offence under this law. The right to treatment requires the state to guarantee that adequate, good quality proper medical, forensic, and mental health services are accessible to the victims without any discrimination (Ministry of Health and Family Welfare (MOHFW), 2014). An excellent health care service requires professionals who can treat injuries, do medico-legal examinations, collect evidence, provide prophylaxis, test for sexually transmitted infections, administer emergency contraception, and give psychosocial support to the victims.\n\nThe Indian health care delivery system is constituted in such a way that most of the reported sexual assault cases are managed by the emergency departments of the hospitals that do not have health care professionals with specialised training in forensic science (Saxena et al., 2015). The situation is similar in state-run as well as in private hospitals, except for certain medical colleges. The emergency team consists of doctors, nurses, and paramedics, in which doctors have the minimum qualification of Bachelor of Medicine and Bachelor of Surgery (MBBS). The curriculum of MBBS contains a para-clinical subject of ‘forensic medicine including toxicology’, which is a stand-alone exposure to the field of forensic sciences. Meanwhile, nurses lack forensic training in any form, as the syllabus of Bachelor of Nursing (BSN) does not have forensic science as a subject. However, nurses have a very significant role in the field of emergency and trauma care, toxicology, crime scene investigation, and correctional settings (Henninger et al., 2020). Strengthening forensic nursing education in the country is critical as the crimes against the women and children are increasing. There is a wider scope for forensic nursing in India and the nurses can take roles in medico legal investigations and evidence collection (Lynch, 2011; Renjith et al., 2016).\n\nMedico-legal care of the victims of sexual assault is very challenging as it involves medical, legal, and ethical aspects. The curriculum of BSc Nursing, as well as General Nursing and Midwifery, does not deal with forensic clinical examination and the collection and preservation of evidence. Unlike the sexual assault referral centres (SARC) in the global scenario, India does not have such designated centres, and the victims are taken care of by the health care centres and state-run hospitals. The nurse in these health care centres needs to be competent enough to handle the medico-legal cases that require specific knowledge and skills (Alsaif et al., 2014). Training on a wide range of issues is important for the nursing staff handling medico-legal cases (Zerbo et al., 2018). These include positioning and examination of the client, obtaining high-quality specimens, identifying and reporting genital-anal injuries after sexual assault, comparing the injuries of consensual and non-consensual intercourse, identifying and collecting traces from the fingernail and other materials for the DNA analysis of the aggressor and employing DNA evidence in sexual offence cases to aid the identification of suspects, movement of forensic evidence and chain of custody, analysing and preventing drug-facilitated crimes against women, usage of photo colposcopy to identify hymenal transections and other injuries in children and adult females, simulation, forensic photography, bite-mark identification, handling vicarious trauma and mindfulness based interventions for better coping. Peer support is also essential for nurses to be able to cope with the demands of working in such situations (Drake and Adams, 2015; Gharedaghi et al., 2018; Yesodharan et al., 2018, 2021; Rodriguez et al., 2019; Usman et al., 2019; Yassa and Badea, 2019; McAllister and Vennum, 2021; Zweig et al., 2021).\n\nA lacuna is identified in the literature related to forensic examination of sexual assault victims. A few studies were identified which assess the knowledge of nurses and student nurses in forensic medical examination. However, no studies were reported from India or Asia regarding the knowledge and skills of nurses in forensic examination and evidence collection.\n\nThe attribute service quality model by Haywood-Farmer was adopted as the conceptual framework for this study (Haywood-Farmer, 1988). This model states that a service organisation is termed high quality if it consistently meets customer preferences and expectations. The separation of attributes into various groups is the first step towards developing service quality. The three types of quality attributes of health care services are physical and process components, behavioural elements, and elements of professional judgment.\n\nThe proposed research is designated to determine the level of the nurse’s competence and knowledge in the examination and management of sexual assault and investigate the effectiveness of multicomponent training in improving knowledge and skill. Implementation of multicomponent training enables nurses to communicate efficiently, examine precisely, collect and preserve evidence accurately and present it properly before the court of law.\n\nThe increase in the number of sexual crimes against women and children, stringent guidelines and protocols issued by the government, the influence of mass media, and increased social pressure demands health care professionals to be more accurate and sensitive in handling cases of sexual assault. The findings of the study will help to develop a protocol for nurses, and the same can be utilised as a model guideline for nurses in India. The findings of the study are expected to initiate the sexual assault nurse examiners network (SANE network) in the Udupi district, which can be adopted to other districts.\n\n\n\ni. Develop a valid and reliable instrument to measure the knowledge regarding SAE among nurses\n\nii. Develop a SANE-GP module using a three-stage Delphi method\n\niii. Explore the effectiveness of SANE-GP on the knowledge and skill regarding SAE.\n\niv. Develop a platform for the SANE network in the country\n\n\nProtocol\n\nThe proposed study adopts a cross-sectional survey design in Phase-I and an experimental design to meet the objectives in Phase-II. The study also uses a Delphi technique for the development of a module for the SANE-GP.\n\nPhase-1\n\nObjective 1: A questionnaire on nurses’ knowledge about sexual assault examination-(KQSANE-I) will be developed based on the Robert F DeVellis method of tool development (DeVellis, 2016), which includes item development, item pooling, content adequacy assessment, clarification of the items, refining the items, the administration of questionnaire (empirical testing after determining the scale for items and adequate sample size), item analysis, reliability assessment, and construct validity. The administration of the questionnaire will be conducted through a survey, and the data will be collected from 450 participants. Skill questionnaires and practice checklists will be developed along with the development of the SANE-GP module.\n\nObjective 2: Delphi techniques of data collection with experts in the field of forensic science to develop a SANE-GP module for nurses.\n\nPhase-2\n\nObjective 3: The study proposes to have one group with a before and after experimental design with three follow-ups to evaluate the effectiveness of SANE-GP among nurses working in the hospital.\n\nPhase-I of the study will be carried out amongst the registered nurses from private and state-run hospitals from Udupi and Dakshina Kannada districts of Karnataka, India. Participants will be included from both genders. A three-stage ‘Delphi’ technique will also be initiated to develop the module for Phase-II. The experts are from the field of forensic medicine and forensic nursing from the national and international arena. In the second phase, 74 nurses from a selected tertiary care hospital will be recruited for the training of SANE-GP and subsequent assessment of the effectiveness of the program.\n\nIn Phase-I, the item pool, which is developed after reviewing the current literature and validated with the experts, will be administered to 450 registered nurses who are willing to participate (see Extended data, Yesodharan et al., 2022a). The sampling technique adopted is purposive sampling. For the development of the module for SANE-GP, Delphi technique data collection will be conducted in three stages. Each stage of Delphi will have seven experts from the field of forensic medicine and forensic nursing. A five-step process will be utilized for the selection of the panel members: i) review the literature and make a list of potential experts based on the recent work in sexual assault examination, ii) check citation index for number of citations, iii) evaluate each expert’ work and grade them on a scale of three, iv) present the potential experts’ work to the Dissertation Advisory Committee (DAC) and develop and final list of experts, v) contact each expert through mail or telephone and explain the purpose with an invitation to participate. A personalised email invitation will be sent to the experts agreed to participate in the study.\n\nThe sample size is calculated for Phase-I based on the item to response ratio of 1:10 given by Schwab (1980). The KQSANE-I is planned to have a minimum of 44 items, hence, the sample size calculated for Phase-I is 450. For Delphi, seven experts will be recruited for participating in the study. The sample size calculated for Phase-II is based on the time series analysis using the formula n = [Z1-α/2 + Z1-β]2 σ2 [1 + (m − 1)ρ]/md2 (where, n is the sample size, Z1-α/2 at α = 0.05 = 1.96, Z1-β at 80% power = 0.84, σ = standard deviation (23.75), ‘m’= number of follow ups (3), ‘ρ’ = intraclass correlation (0.4), ‘d’ = clinically significant difference (7)). The sample size is calculated with an anticipatory non-response rate of 25% which is 74. The participants will be recruited through purposive sampling for SANE-GP.\n\nStudy participants for Phase-I: Registered nurses working in selected hospitals will be included in the study. The hospitals will be selected based on the convenience of the researcher.\n\nFor Delphi technique: Five to seven experts in the field of forensic science and forensic nursing will be selected.\n\nStudy participant for Phase-II: Female registered nurses from 22 to 45 years working in a tertiary care hospital will be included in the study.\n\nPhase-I: Delphi method to finalise the content of the SANE-GP training module. Permission from the respective hospital management will be obtained before the advertisement for the recruitment of nurses to the study. The study process will be informed in detail through an information sheet, and informed consent will be obtained from the participants during Phase-I. The researcher will be present throughout the data collection. Phase-I of the study also contains the development of a training module; a Delphi method will be used to finalise the content of the SANE-GP training module.\n\nThe researchers will do a literature search and prepare the initial draft of the module and the knowledgeable subject experts in DAC will assess the readability of the initial draft. Revisions will be made on the draft module based on their suggestions. When the expert panel list is finalized, a discussion forum will be setup on an online platform by keeping the details of the experts anonymous. The draft module and the other the questionnaire will be made available to the experts either through email or uploading to the online forum. A stage one of Delphi will be initiated and the experts will go through the draft module and assess based on questionnaire. The questionnaire involves items such as whether the content is worded correctly, whether the content is relevant, and whether the content is adequate. All the items have to be graded using a four-point Likert rating scale. The experts will have one-month time to the overall assessment of the draft module. When the responses (Scores and comments) from all the panel experts are available then it will be scrutinised by the researchers and identifies the common and conflicting viewpoints. The draft module will be revised based on the suggestions by the expert panel. The stage one of the Delphi ends with preparation for the stage two. In the stage two of Delphi the scores and the comments from the experts are mailed to the experts and the experts are encouraged to revise their earlier views in light of the replies of other members of the group. The experts will grade the draft module based on the items in the questionnaire and the disagreements will be resolved by discussing and negotiating with the experts. The draft module will be further revised and move forward for the final stage of Delphi. The process will be repeated until all the panellists in the Delphi reach a consensus. A thematic framework will be used for the analysis of the information collected through the Delphi method.\n\nPhase-II: The effectiveness of the SANE-GP will be assessed through administering KQSANE-I. A pretest will be conducted on day one, and an immediate post-test will be conducted after the intervention. Follow-ups will be conducted on the 8th, 16th, and 24th week after SANE-GP. The skill of the participants in performing SAE will be assessed after the completion of each section of the module through a skill checklist. After the implementation of SANE-GP, the participants will be randomly picked for assisting the forensic expert whenever a sexual assault victim is coming to the hospital for examination. The implementation of the SANE-GP will be assessed by assessing the practice of the nurses by the forensic expert (outcome assessor) through a checklist.\n\nTool 1: Demographic Proforma\n\nDemographic proforma includes items such as age, gender, occupation, highest education, years of experience, specialty, and previous experience in SAE.\n\nTool 2: KQSANE-I\n\nThis will be used for assessing the knowledge of participants regarding the sexual assault examination. The tool will be developed in Phase-I after completing all tool development steps, namely, item development, item pooling, content adequacy assessment (clarification of the items and refining the items), the administration of the questionnaire (empirical testing after determining the scale for items and adequate sample size), item analysis, reliability assessment and assessing construct validity.\n\nThe SANE-GP will have multiple teaching-learning activities given to the participants for a period of one week. Each session will last for 45 min and seven sessions will be conducted per day. The content of each module and the order of the sessions also will be finalized after the third stage of the Delphi. Additional modules will be also added based on the suggestions of the expert panel. The details of the modules which will be prepared and send to the panel experts are mentioned below.\n\n1. Overview of forensic and sexual assault examination\n\ni. History of medico-legal examination\n\nii. The constitution of India and human rights\n\niii. Criminal behaviour and criminal body language\n\n2. Female and male genitalia\n\n3. Medico-legal history taking\n\ni. Communication and soft skill development (for caring of victims)\n\nii. Sexual abuse against children\n\niii. Sexual violence against transgendered persons, intersex persons, and persons with alternative sexual orientations (ASO)\n\niv. Sexual violence against elderly\n\nv. Prerequisites for history collection and examination\n\n4. Observing and assessing physical examination findings\n\ni. Local examination of genital parts and other orifices\n\n5. Medico-legal evidence collection\n\ni. Informed consent\n\nii. Evidence and types of evidence\n\niii. Crime scene investigation\n\niv. Sexual assault forensic examination (SAFE) kit\n\nv. Body evidence and anogenital evidence collection\n\nvi. Collection and packing, and preservation of material evidence from the survivor\n\nvii. A chain of custody\n\n6. Forensic photography, colposcopy, and other digital evidence\n\ni. Cameras and accessories\n\n7. Medical management of cases with sexual violence\n\ni. Standard operating protocol\n\n8. Sexually transmitted infection testing and prophylaxis\n\n9. Pregnancy testing and prophylaxis\n\n10. Medico-legal documentation\n\ni. Examination of injuries and intimation to police\n\n11. Legal consideration and judicial proceedings\n\ni. The Criminal Law Amendment Act 2013\n\nii. The Indian Evidence Act 1872\n\niii. The CrPC and the Indian Penal Code\n\niv. The Protection of Children from Sexual Offences Act, 2012 (POCSO Act)\n\nv. Legal responsibilities of health professionals\n\n12. Community collaboration\n\ni. Dealing with police and judiciary and child welfare committee\n\nii. Dealing with public and mass media\n\n13. The psychosocial care of victims and family members\n\n14. Networking of SANE nurses\n\nThe SANE-GP also includes practical activities such as case discussions, discussion of supreme court and trial court verdicts, communication and soft skills demonstrations, documentation of injuries, mock case examinations, simulations, dummy examination, and preparation of slides with the specimen collected from the vaginal vault and other orifices using swabs.\n\nInstitutional Ethics Committee of Kasturba Hospital and Kasturba Medical College Manipal, Karnataka, India, approved the proposed study through wide reference No 653/2018. Complete information about the study will be given to the participants through an information sheet and written informed consent will be obtained (see Extended data, Yesodharan et al., 2022b, c, d).\n\nThe current study is aimed at assessing the knowledge, skill, and practice of nurses regarding sexual assault examination. The knowledge will be assessed before SANE-GP, and three follow-ups will be conducted on the 8th, 16th, and 24th week after SANE-GP. The researchers will meet the participants in their work area and administer the questionnaires physically. The skill will be assessed immediately after the completion of each module in the SANE-GP whereas the practice of the participants will be assessed randomly in the presence of the forensic expert from the research setting using a practice checklist. Once the participants complete the SANE-GP, they will be enrolled in an association registered under the Societies Registration Act, 1860. This platform will be used for the communication and future development of SANE programs in India.\n\nThe researchers will use statistical software by IBM (IBM SPSS Statistics, RRID:SCR_019096), SPSS statistics 26 (Armonk, NY: IBM Corp) for the analysis. In Phase-I, the data will be summarised using descriptive statistics and will be presented in a summary table. The knowledge score will be assessed four times (one before SANE-GP and three follow-ups), and the scores will be compared and analysed with the help of time-series analysis. The skill of the participants will be assessed after the completion of each module, and the score will be described in tables. The practice of the random participants will be assessed and described.\n\nResults will be disseminated via presentations at appropriate scientific conferences and meeting of professional bodies. The findings will also be published in peer-reviewed journals, professional and institutional repositories etc. The result will be discussed with the Governmental bodies and other stakeholders for improvement of process in medico-legal evidence collection.\n\nThe study team is in the process of recruitment of participants for the phase I of the study.\n\n\nDiscussion\n\nForensic examination of the sexually assaulted individual has multifaceted challenges ranging from therapeutic to legal. To take care of such individuals, the forensic team including the nurse should be competent enough to deal with the challenges emerging out. A forensic examination is not everybody’s business; keeping it open to all may hamper the evidence and affect the admissibility of the evidence in the court of law, helping the culprits to escape. An unprofessional and unskilled way of collecting evidence can make victims re-experience the trauma and lead to physiological and psychological distress.\n\nIn India, the SAE is currently done by a forensic expert or a clinician/physician with the assistance of a nurse. The nursing professional who is assisting is not trained or taught forensic evidence collection. Moreover, the communication with the legal authorities and the victim may be affected if the nurse is not trained to deal with those types of cases. The Indian medico-legal system only allows doctors to conduct the medico-legal examination, unlike the United States of America or the United Kingdom, which allows competent and licensed Sexual Assault Nurse Examiners (SANE) to do so. We do not have any specialised course or any accrediting agency to certify the nurses to do the sexual assault examination, which compromises the care of the victim, collection of evidence, and reporting of facts to the court.\n\nThe current research is important because for the first time in India, it proposes a minimum requirement for the training of nurses regarding sexual assault examination. The research also proposes a guideline for the practising of sexual assault examination by the nurses. The specific role of the trained nurse increases the accountability and responsibility in the evidence collection and care of the victims. The study also proposes a network of forensic nurses so that all cases reporting in any hospitals, nursing homes, and clinics will get the service of the trained nurse irrespective of their current employment.\n\nA study by Cunha et al. (2016) evaluated the knowledge of nursing students over forensic practices using a KQFNP, which showed that there was an insufficient knowledge over the practical aspects of forensic nursing (36.3%). The study highlighted the importance of enhancing the training of the nursing students about the forensic nursing aspects allowing them to adopt good clinical practices.\n\nCucu et al. (2014) conducted a descriptive study in the transversal approach and identified and described the knowledge, experiences, and training needs of nurses related to forensic patients. In total, 30 nurses from the emergency department were assessed with a 53-item self-administered questionnaire. The results of the study based on correct answer scores showed that a small group of the participants (13%) performed poorly (less than 5), and a majority (63%) did relatively well (6-10) than the former on a 17-item knowledge questionnaire. The study results also revealed that all the nurses agreed (100%) that they required solid training on forensic topics. The participants also ranked ten specific topics areas concerning forensic patient care, and the major five were forensic patient communication (4.7 ± 0.88), the legal aspect of forensic medicine (4.67 ±0.80), violence (4.57 ±0.97), traffic accidents (4.53 ±0.86), and sexual assault (4.43 ±1.07). The survey concluded that forensic training is desirable and needed among emergency department nurses as an assurance to render appropriate care together with proper management of medico-legal evidence and advocate for the patient rights.\n\nFeizi Nazarloo et al. (2017), through a cross-sectional study, revealed that emergency nurses had the least knowledge in the collection and protection of forensic evidence. Among the 195 participants, 95.4% had no formal training in managing the forensic patients, 92.3% had stated that there was no formal written guideline in caring for forensic patients, and 95.9% had educational needs for managing the forensic patients. The overall knowledge status about forensic nursing is low in emergency nurses (44.13%), which emphasise the need for specialised education and training in forensic nursing.\n\nAlthough the study aims to develop the SANE program, it requires widespread acceptance across the hospitals in the country. The absence of policy changes and revision of existing guidelines are a few challenges that can block the nationwide implementation of SANE programs. Sustainable planning is also needed for the development of a platform that connects the SANEs across the country. The study is planned as an experimental design and does not involve any randomisation or control.\n\n\nConclusions\n\nThe results from the proposed study are expected to help the nurses working in emergency departments, and outpatient departments and sexual assault referral centres to become knowledgeable, skilful, flexible, non-judgmental, empathetic and understanding, supportive, and resilient. It will also help them to demonstrate adequate coping skills, collaborate, and support other team members.\n\n\nData availability\n\nNo underlying data are associated with this article.\n\nFigshare: KQSANE.pdf. https://doi.org/10.6084/m9.figshare.18865136.v2 (Yesodharan et al., 2022a).\n\nThis project contains the following extended data:\n\n- KQSANE.pdf (The item pool for the development of the tool KQSANE-I)\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).\n\nFighsare: Informed Consent.doc. https://doi.org/10.6084/m9.figshare.18865130.v3 (Yesodharan et al., 2022b).\n\nFigshare: Participant Information Sheet-Phase 1.doc. https://doi.org/10.6084/m9.figshare.18865139.v2 (Yesodharan et al., 2022c).\n\nFigshare: Participant Information Sheet-Phase 2.doc. https://doi.org/10.6084/m9.figshare.18865133.v2 (Yesodharan et al., 2022d).\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\nJaithlia A, Maheshwari A: The Agonizing Pace of the Indian Judiciary. Jurist. 2020. May. Reference Source\n\nAlsaif DM, et al.: Forensic experience of Saudi nurses; an emerging need for forensic qualifications. J. Forensic Legal Med. 2014; 27: 13–16. PubMed Abstract | Publisher Full Text\n\nCucu A, et al.: Forensic Nursing Emergency Care. Rom. J. Leg. Med. 2014; 22(2): 133–136. Publisher Full Text\n\nCunha M, Libório R, Coelho M: Knowledge Questionnaire over Forensics Nursing Practices. Procedia. Soc. Behav. Sci. 2016; 217: 1089–1097. Publisher Full Text\n\nDeVellis RF: Scale development: Theory and applications. 4th ed.Chapel Hill, USA: Sage Publications; 2016.\n\nDrake SA, Adams NL: Three Forensic Nursing Science Simulations. Clin. Simul. Nurs. 2015; 11(3): 194–198. Publisher Full Text\n\nFeizi Nazarloo L, et al.: Emergency Department Nurses’s Knowledge about Forensic Nursing. J. Holist. Nurs. Midwifery. 2017; 27(3): 27–36. Publisher Full Text\n\nFlecha R: Second-Order Sexual Harassment: Violence Against the Silence Breakers Who Support the Victims. Violence Against Women. 2021; 27(11): 1980–1999. PubMed Abstract | Publisher Full Text\n\nGharedaghi F, et al.: Drug-facilitated crime caused by drinks or foods. Egypt. J. Forensic Sci. 2018; 8(1): 68. Publisher Full Text\n\nHaywood-Farmer J: A Conceptual Model of Service Quality. Int. J. Oper. Prod. Manag. 1988; 8(6): 19–29. Publisher Full Text\n\nHenninger AL, et al.: Reporting Sexual Assault: Survivors’ Satisfaction With Sexual Assault Response Personnel. Violence Against Women. 2020; 26(11): 1362–1382. Publisher Full Text\n\nJennings WG, Powers RA, Perez NM: A Review of the Effects of the Violence Against Women Act on Law Enforcement. Violence Against Women. 2021; 27(1): 69–83. PubMed Abstract | Publisher Full Text\n\nLynch VA: Forensic nursing science: Global strategies in health and justice. Egypt. J. Forensic Sci. 2011; 1(2): 69–76. Publisher Full Text\n\nMaier SL: “I Have Heard Horrible Stories …”: Rape Victim Advocates’ Perceptions of the Revictimization of Rape Victims by the Police and Medical System. Violence Against Women. 2008; 14(7): 786–808. PubMed Abstract | Publisher Full Text\n\nMaier SL: Sexual assault nurse examiners’ perceptions of the revictimization of rape victims. J. Interpers. Violence. 2012; 27(2): 287–315. PubMed Abstract | Publisher Full Text\n\nMcAllister P, Vennum A: Sexual Violence and Mental Health: An Analysis of the Mediating Role of Self-Compassion Using a Feminist Lens. Violence Against Women. 2021. PubMed Abstract | Publisher Full Text\n\nMinistry of Health and Family Welfare (MOHFW): Guidelines & Protocols. Medico-legal care for survivors/victims of sexual violence.2014. (Accessed: 26 August 2017). Reference Source\n\nMurshid NS, Bowen EA: A Trauma-Informed Analysis of the Violence Against Women Act’s Provisions for Undocumented Immigrant Women. Violence Against Women. 2018; 24(13): 1540–1556. PubMed Abstract | Publisher Full Text\n\nNational Crime Records Bureau (NCRB): Crime in India 2019 Volume 1. New Delhi: 2019. Reference Source\n\nRelyea M, Ullman SE: Predicting Sexual Assault Revictimization in a Longitudinal Sample of Women Survivors: Variation by Type of Assault. Violence Against Women. 2017; 23(12): 1462–1483. 2016/08/23. Publisher Full Text\n\nRenjith V, et al.: Forensic nursing. Indian J. Forensic Med. Toxicol. 2016; 10(2): 159–162. Publisher Full Text\n\nRodriguez JJRB, et al.: Integrating presumptive and confirmatory semen tests into DNA profiling of sexual assault evidence: a Philippine example. Egypt. J. Forensic Sci. 2019; 9(1): 45. Publisher Full Text\n\nRukmini S: Marital and other rapes grossly under-reported. The Hindu. 2014. Reference Source\n\nSaxena A, et al.: Pattern of medico-legal cases in the casualty Department of a teaching hospital, Bareilly, Uttar-Pradesh. J. Indian Acad. Forensic Med. 2015; 37(4): 338. Publisher Full Text\n\nSchwab DP: Construct validity in organization behavior. Barry MS, Larry LC, editors. Research in organizational behavior. Greenwich, CT: JAI; 1980; pp. 3–43.\n\nShanmugam R: Informatics About Fear to Report Rapes Using Bumped-up Poisson Model. Am. J. Biostat. 2013; 3(1): 17–29. Publisher Full Text\n\nUsman M, et al.: Forensic toxicological analysis of hair: a review. Egypt. J. Forensic Sci. 2019; 9(1): 17. Publisher Full Text\n\nYassa HA, Badea ST: Patterns of drug abuse in Upper Egypt: cause or result of violence?. Egypt. J. Forensic Sci. 2019; 9(1): 14. Publisher Full Text\n\nYesodharan R, et al.: Forensic clinical photography: A game changer in medicolegal investigation and forensic science. Indian J. Forensic Med. Toxicol. 2018; 12(2): 262–266. Publisher Full Text\n\nYesodharan R, et al.: Looking through the lens of a sexual assault examiner: novel trends and approaches in forensic photography. Egypt. J. Forensic Sci. 2021; 11(1): 27. Publisher Full Text\n\nYesodharan R, et al.: KQSANE.pdf. figshare. Online resource.2022a. Publisher Full Text\n\nYesodharan R, et al.: INFORMED CONSENT.doc. figshare. Online resource.2022b. Publisher Full Text\n\nYesodharan R, et al.: PATICIPANT INFORMATION SHEET- PHASE 1.doc. figshare. Online resource.2022c. Publisher Full Text\n\nYesodharan R, et al.: PARTICIPANT INFORMATION SHEET -PHASE-2.doc. figshare. Online resource.2022d. Publisher Full Text\n\nZerbo S, et al.: Medico legal procedures related to sexual assault: a 10-year retrospective experience of a Daphne protocol application. Egypt. J. Forensic Sci. 2018; 8(1): 4. Publisher Full Text\n\nZweig J, et al.: Community Approaches to Sexual Assault: VAWA’s Role and Survivors’ Experiences. Violence Against Women. 2021; 27(1): 30–51. PubMed Abstract | Publisher Full Text"
}
|
[
{
"id": "135825",
"date": "12 May 2022",
"name": "Udara Dilrukshi Senarathne",
"expertise": [
"Reviewer Expertise Pathology",
"Forensic Medicine",
"Sexual Abuse Examination"
],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 protocol addresses an unmet need in the health sector, especially in Asian countries. Although the nurses are pretty involved in the emergency department patient care and ward patient care, they do not routinely receive training on examining a sexual assault victim. Such training is vital as it is essential to obtain useful evidence, administer appropriate treatment measures, educate the victim on certain aspects, and, most importantly, avoid re-traumatizing the victim during the healthcare processes.\n\nThis study protocol addresses all necessary steps in developing such a training program and auditing its outcome.\n\nSince this is the first time such a program would be introduced to the system, I suggest adding a qualitative interview-based section to Phase II in assessing the effectiveness of the SANE-GP by interviewing randomly selected participants using open/close-ended questions to enrich the study outcome.\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": "8326",
"date": "17 Jun 2022",
"name": "RENJULAL YESODHARAN",
"role": "Author Response",
"response": "Dear Dr Udara, Thank you for reviewing our work. We will look into the suggestions you have given. A qualitative study will be initiated after completing this project."
}
]
},
{
"id": "138252",
"date": "28 Jun 2022",
"name": "Amita Pitre",
"expertise": [
"Reviewer Expertise I have a masters in Health Sciences",
"and extensive work in the response of the health system to sexual assault. I am the Lead Specialist",
"Gender Justice at Oxfam India. I have done extensive work in gender based violence. I have several publications linked to the same which are accessible on my Academia.edu and Researchgate sites. I was among the first ones to talk about the need for standard protocols and gender training including technical competency training for doctors in examination of sexual assault survivors. I have written extensively on survivor centric approaches to care."
],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis appears to be an article based on study design of a prospective study. The rationale of the study needs to specifically mention why they are at all considering the training of nurses given that it is not legal as yet. The rationale also needs to at least flag why SANEs are important.\nI have addressed below what else needs to be included to provide a convincing rationale. Summarising here:\n\nDatasets to be used are not completely clear. Only quantitative and statistical data sets seem to have been provided or listed to be used. There will be substantial qualitative information gathered or needs to be gathered from SANE's experiences and if possible those of women; what improved what did not; what are the challenges faced by SANEs; were they given adequate space in a highly patriarchal, hierarchical set up where doctors have the legal privilege to examine. More details below.\n\nThe literature review can draw more upon the Indian scenario if possible. For example the Henninger citation does not talk about the Indian scenario as far as I can see. So why are Nurses a crucial ally in examination of sexual assault survivors and why they are well placed to be trained as SANEs can be better illustrated from Indian experience if possible. Or then state global evidence shows this and scenario in India is not likely to be different given the important role of nurses in health care provision, including in emergencies.\n\nIntroduction and Research Gap\nWhile providing the rationale of the study and the introduction, you can mention here that law expects a woman to be examiner or given first preference and survivors also find it more acceptable. For gender sensitive care it makes sense to have women examiners. Nurses are almost universally women in India and easier to access than women doctors.\n\nInclusion criteria:\nHere you mention in phase 2 female nurses will be enrolled and earlier in the methodology you mention persons from both genders would be included. It would be good to explain the difference and how and why this has been devised.\n\nSANE Grounding Program (SANE-GP); point 2\nIdeally the spectrum of human genitalia including intersex needs to be added here- not just male and female. Intersex and transpersons also face substantial risk of sexual assault.\n\nSANE Grounding Program (SANE-GP); point 2\nPerspective in the entire paper is very medical and may risk objectifying the survivor. E.g. medical management of case instead of provision of health care to survivor. However this should not amount to language dressing but needs to permeate the intent and roll out of study and will then naturally flow in the paper.\n\nEthical considerations:\nThis is very inadequate for such a sensitive topic. Please mention specific anticipated ethical concerns such as trauma that potential participants may face, how you will prepare them for it, what care will be available, what will be done to prevent it; several ethical concerns related to mandatory reporting to police- sometimes survivors forego medical treatment to escape reporting - how will that be taken care of; legally nurses are not allowed to practice and in future after the research they may not be allowed to - how will their anticipations be managed; how will the research prevent duplication of examination in case the forensic expert is supervising; how will the survivor be informed about their inclusion in a research- what safeguards, informed consent available to them etc will nurses be able to testify in court etc. These need to be robustly stated and potential resolutions and risks noted.\n\nCompetency of nurses:\nStrengths of nurses need also to be listed. Why nurses? Women, known to be more sensitive to patients, better at developing rapport with the survivor, would put her at ease, better acceptance of examination, better history taking since she is a woman and easier for women to open up etc. instead of only focusing on building the competency of nurses, state why SANE program would be better than simply training more doctors.\n\nAbout knowledge of nurses in forensic examination- It has been stated that they do not have the subject in their course and they are not legally allowed to examine. Therefore this statement about their lack of knowledge needs to be qualified with this information.\n\nInstead of only focusing on lacunae in nursing training, need to highlight how training of nurses is also in the interest of women, health system, better delivery of service. As women they will also be able to better empathise and understand the trauma of the woman.\n\nMethodology\n\nMethodology needs to also include feedback from survivors. It could also have a control group- cohort with SANE involvement and without.\n\nDiscussion\nThe discussion section is very limited and focused on the need to train nurses. However, it needs more depth about the shortcomings of a doctor led model, the critique of the current system, lack of knowledge of doctors themselves and shoddy services despite having it in medical curriculum and specific training, dissonance between doctors actually invited to examine (gynaecologists and women doctors) vs those who are trained (forensics).\n\nThere is ample literature on current limitations of the medical model and system of examination- most recent by Lakshmi Lingam, Sunita Bandewar and Sita Mammudipudi from Tata Institute of Social Sciences for the ICMR; Human Rights Watch report of a few years back. Multiple writings including my own. All these need to be present from literature review stage onwards and brought together in discussion. So limitations of current model, strengths of nurses doing exam, need for their training and how it may improve the system, legal limitations in doing so, future challenges all need to come together in discussion.\n\nConclusion:\nAgain this is very limiting. The aim is not to enlighten nurses but to add value for the survivors medico-legal exam, more sensitivity, accuracy, etc etc. The heart of the matter is not captured in the discussion, conclusions and the entire approach of the research is to educate nurses. Needs above changes.\n\nRegarding additional literature, here are two studies (one of which I technically reviewed and gave inputs to) and one letter to the editor (I co-authored), which is useful to draw upon how the system for medico-legal examination in India is still poorly developed.\nEnhancing the Quality of Response of the Health Care System to Sexual Assault. Dignity on Trial - India’s Need for Sound Standards for Conducting and Interpreting Forensic Examinations of Rape Survivors Doctors in India continue to traumatise rape survivors with the two-finger test Need for gender sensitive health system responses to violence against women and children\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": [
{
"c_id": "9116",
"date": "09 Jan 2023",
"name": "RENJULAL YESODHARAN",
"role": "Author Response",
"response": "\"This appears to be an article based on study design of a prospective study. The rationale of the study needs to specifically mention why they are at all considering the training of nurses given that it is not legal as yet. The rationale also needs to at least flag why SANEs are important. I have addressed below what else needs to be included to provide a convincing rationale. Summarising here:\" Thank you for reviewing the protocol and giving your valuable suggestions. Modifications are made accordingly \"Datasets to be used are not completely clear. Only quantitative and statistical data sets seem to have been provided or listed to be used. There will be substantial qualitative information gathered or needs to be gathered from SANE's experiences and if possible those of women; what improved what did not; what are the challenges faced by SANEs; were they given adequate space in a highly patriarchal, hierarchical set up where doctors have the legal privilege to examine. More details below.\" Thank you for your suggestion. The proposed study aimed to collect quantitative data set and implementation of the SANE-GP. The researchers are planning to conduct a qualitative study after the implementation of the program. The future studies will definitely involve the SANE experiences and experiences of survivors. \"The literature review can draw more upon the Indian scenario if possible. For example the Henninger citation does not talk about the Indian scenario as far as I can see. So why are Nurses a crucial ally in examination of sexual assault survivors and why they are well placed to be trained as SANEs can be better illustrated from Indian experience if possible. Or then state global evidence shows this and scenario in India is not likely to be different given the important role of nurses in health care provision, including in emergencies.\" Thank you for the suggestion. Unfortunately, there are not much literature available regarding Indian Scenario. Studies related to Sexual Assault Examinations are very limited. The literature suggested by the reviewers are included in the literature review and discussion. \"Introduction and Research Gap: While providing the rationale of the study and the introduction, you can mention here that law expects a woman to be examiner or given first preference and survivors also find it more acceptable. For gender sensitive care it makes sense to have women examiners. Nurses are almost universally women in India and easier to access than women doctors\" Thank you for the suggestions. Authors completely agree to this point and modifications are made accordingly \"Inclusion criteria: Here you mention in phase 2 female nurses will be enrolled and earlier in the methodology you mention persons from both genders would be included. It would be good to explain the difference and how and why this has been devised.\" The entire study is divided into two phases. The first Phase is for the development of the tool. The developed tool can be administered among females as well as males for future research also. Therefore, the researchers included male participants also. Phase I has no influence on Phase II of the study. Whereas, the Phase II of the study is the implementation of SANE-GP and it is limited to female participants alone. These two phases of the study are standalone. \"SANE Grounding Program (SANE-GP); point 2 Ideally the spectrum of human genitalia including intersex needs to be added here- not just male and female. Intersex and transpersons also face substantial risk of sexual assault.\" Module II is dealing with the anatomy and physiology of female and male genitalia. The heading of this module is changed to “Male, female, intersex and ambiguous genitalia” The modules or the content of the modules are yet to be finalized through Delphi method. Panel experts will evaluate the modules and module headings. Under module III. The issues against Sexual violence against transgendered persons, intersex persons, and persons with alternative sexual orientations (ASO) are also detailed. \"SANE Grounding Program (SANE-GP); point 2 Perspective in the entire paper is very medical and may risk objectifying the survivor. E.g. medical management of case instead of provision of health care to survivor. However this should not amount to language dressing but needs to permeate the intent and roll out of study and will then naturally flow in the paper.\" We completely agree with the reviewer to the point about objectifying the survivor modifications were made accordingly \"Ethical considerations: This is very inadequate for such a sensitive topic. Please mention specific anticipated ethical concerns such as trauma that potential participants may face, how you will prepare them for it, what care will be available, what will be done to prevent it; several ethical concerns related to mandatory reporting to police- sometimes survivors forego medical treatment to escape reporting - how will that be taken care of; legally nurses are not allowed to practice and in future after the research they may not be allowed to - how will their anticipations be managed; how will the research prevent duplication of examination in case the forensic expert is supervising; how will the survivor be informed about their inclusion in a research- what safeguards, informed consent available to them etc will nurses be able to testify in court etc. These need to be robustly stated and potential resolutions and risks noted.\" The study does not involve any sexual assault survivors and the participants of the study are nurses. The detailed ethical issues are addressed in the documents submitted to the Ethics committee and the participant information sheet and informed consent are published in the extended data. The aim of the study is to make nurses compete to handle the medico-legal care of the survivors rather than examining them. Examination and documentation are only one aspect of the medico-legal care of the survivors. The nurses are the first person who receives the patient in the emergency department or in any other area concerned with medical care. Medicolegal care excluding nurses is not feasible and they are considered an integral part of it. The nurses trained will be part of the forensic team and they will handle the client in a more professional way along with the Forensic expert. The nurses perform no such examinations independently until the law legally permits it. Hence, the ethical concerns associated with the examination by the nurses are out of the scope of the study. \"Competency of nurses: Strengths of nurses need also to be listed. Why nurses? Women, known to be more sensitive to patients, better at developing rapport with the survivor, would put her at ease, better acceptance of examination, better history taking since she is a woman and easier for women to open up etc. instead of only focusing on building the competency of nurses, state why SANE program would be better than simply training more doctors.\" Thank you for the suggestions. Strengths of the nurses are also included in the manuscript \"About knowledge of nurses in forensic examination- It has been stated that they do not have the subject in their course and they are not legally allowed to examine. Therefore this statement about their lack of knowledge needs to be qualified with this information. Instead of only focusing on lacunae in nursing training, need to highlight how training of nurses is also in the interest of women, health system, better delivery of service. As women they will also be able to better empathise and understand the trauma of the woman.\" Thank you for notifying this point, Text is modified as per the suggestions \"Methodology Methodology needs to also include feedback from survivors. It could also have a control group- cohort with SANE involvement and without.\" The study does not involve any survivors hence; feedback is practically not possible. A qualitative study will be conducted after the implementation of the current project, which includes feedback from all the stakeholders. \"Discussion The discussion section is very limited and focused on the need to train nurses. However, it needs more depth about the shortcomings of a doctor led model, the critique of the current system, lack of knowledge of doctors themselves and shoddy services despite having it in medical curriculum and specific training, dissonance between doctors actually invited to examine (gynaecologists and women doctors) vs those who are trained (forensics).There is ample literature on current limitations of the medical model and system of examination- most recent by Lakshmi Lingam, Sunita Bandewar and Sita Mammudipudi from Tata Institute of Social Sciences for the ICMR; Human Rights Watch report of a few years back. Multiple writings including my own. All these need to be present from literature review stage onwards and brought together in discussion. So limitations of current model, strengths of nurses doing exam, need for their training and how it may improve the system, legal limitations in doing so, future challenges all need to come together in discussion.\" The shortcomings of the current model are discussed. The authors included the suggested references in the discussion. The authors thank the reviewer for suggesting the valuable works \"Conclusion: Again this is very limiting. The aim is not to enlighten nurses but to add value for the survivors medico-legal exam, more sensitivity, accuracy, etc etc. The heart of the matter is not captured in the discussion, conclusions and the entire approach of the research is to educate nurses. Needs above changes.\" Conclusions modified accordingly \"Regarding additional literature, here are two studies (one of which I technically reviewed and gave inputs to) and one letter to the editor (I co-authored), which is useful to draw upon how the system for medico-legal examination in India is still poorly developed Enhancing the Quality of Response of the Health Care System to Sexual Assault. Dignity on Trial - India’s Need for Sound Standards for Conducting and Interpreting Forensic Examinations of Rape Survivors Doctors in India continue to traumatise rape survivors with the two-finger test Need for gender sensitive health system responses to violence against women and children\" All the works suggested are included in the manuscript."
}
]
}
] | 1
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https://f1000research.com/articles/11-134
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https://f1000research.com/articles/11-741/v1
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04 Jul 22
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{
"type": "Research Article",
"title": "Neural crest cell-placodal neuron interactions are mediated by Cadherin-7 and N-cadherin during early chick trigeminal ganglion assembly",
"authors": [
"Caroline A. Halmi",
"Chyong-Yi Wu",
"Lisa A. Taneyhill",
"Caroline A. Halmi",
"Chyong-Yi Wu"
],
"abstract": "Background: Arising at distinct positions in the head, the cranial ganglia are crucial for integrating various sensory inputs. The largest of these ganglia is the trigeminal ganglion, which relays pain, touch and temperature information through its three primary nerve branches to the central nervous system. The trigeminal ganglion and its nerves are composed of derivatives of two critical embryonic cell types, neural crest cells and placode cells, that migrate from different anatomical locations, coalesce together, and differentiate to form trigeminal sensory neurons and supporting glia. While the dual cellular origin of the trigeminal ganglion has been known for over 60 years, molecules expressed by neural crest cells and placode cells that regulate initial ganglion assembly remain obscure. Prior studies revealed the importance of cell surface cadherin proteins during early trigeminal gangliogenesis, with Cadherin-7 and neural cadherin (N-cadherin) expressed in neural crest cells and placode cells, respectively. Although cadherins typically interact in a homophilic (i.e., like) fashion, the presence of different cadherins on these intermingling cell populations raises the question as to whether heterophilic cadherin interactions may also be occurring during initial trigeminal ganglion formation, which was the aim of this study. Methods: To assess potential interactions between Cadherin-7 and N-cadherin, we used biochemistry and innovative imaging assays conducted in vitro and in vivo, including in the forming chick trigeminal ganglion. Results: Our data revealed a physical interaction between Cadherin-7 and N-cadherin. Conclusions: These studies identify a new molecular basis by which neural crest cells and placode cells can aggregate in vivo to build the trigeminal ganglion during embryogenesis.",
"keywords": [
"cadherins",
"neural crest cells",
"placode cells",
"trigeminal ganglion",
"chick embryo"
],
"content": "Introduction\n\nCranial ganglia are sensory structures of the peripheral nervous system possessing the cell bodies of the cranial nerves. These ganglia and their associated nerves function in olfaction, taste, hearing, vision, and somatosensation.1–3 The trigeminal ganglion, the largest of the cranial ganglia, contains three sensory branches (ophthalmic, maxillary, and mandibular) that innervate different regions of the face to mediate sensations of pain, touch, and temperature.3–5 During embryonic development, two distinct cell populations, neural crest cells and neurogenic placode cells, intermingle and aggregate to generate the trigeminal ganglion.6–10 These interactions have been studied for over 60 years and reveal that each cell type contributes distinctly to trigeminal ganglion formation, with neural crest cells acting as a scaffold for the integration of placode cell-derived neurons, while placodal neurons aid in the condensation of neural crest cells.7,10,11 Moreover, ablation of either of these cell populations leads to severe defects in trigeminal ganglion development, indicating a reciprocal relationship.7,10,12\n\nPrior studies indicate that intercellular interactions during trigeminal ganglion formation are mediated, in part, by cadherin-based adhesion. Two cadherins, Cadherin-7 and neural cadherin (N-cadherin), are expressed in neural crest cells and placode cells, respectively, during trigeminal gangliogenesis. Expression of Cadherin-7, a type II classical cadherin, was discovered in migratory cranial neural crest cells in the chick embryo over 25 years ago.13 More recent studies of Cadherin-7 protein confirmed previous in situ hybridization findings and noted Cadherin-7 in chick migratory cranial neural crest cells contributing to the trigeminal ganglion.14 Both depletion and overexpession of Cadherin-7 impact the distribution of chick embryonic neural crest cells and placodal neurons, and as such, the overall morphology of the ganglion. N-cadherin, a type I classical cadherin, is present throughout development and has been found in derivatives of the endoderm, mesoderm, and ectoderm.15 Notably, both ectodermal placode cells and their neuronal derivatives express N-cadherin16 in the chick trigeminal ganglion. Knockdown of N-cadherin does not affect initial placode cell ingression and delamination from the ectoderm,16 but leads to increased placodal neuron dispersal during trigeminal gangliogenesis. Conversely, N-cadherin overexpression causes aberrant aggregation of placodal neurons.16 Modulation of N-cadherin levels appears to involve, in part, post-translational mechanisms linked to Slit1-Robo2 signaling in the developing chick trigeminal ganglion,16 but specific details underlying this process are not known.\n\nWhile the ability of cadherins to make homophilic interactions is well understood, cadherins can also make heterophilic (i.e., non-like) connections with other cadherins, either in the same (homotypic) or different (heterotypic) cell types. Observations of heterophilic cadherin interactions have been reported during normal development of the endoderm,17 in establishing synaptic potentials within the hippocampus,18 and during Xenopus gastrulation,19 and are also noted in diseases such as cancer.20 In addition, the atypical cadherins Fat and Dachsous are capable of forming heterodimers between neighboring homotypic cells.21 Collectively, these results support the notion that heterophilic interactions can occur between different types of cadherins during development. While previous studies noted the formation of aggregates from mixtures of N-cadherin- and Cadherin-7-expressing cells in vitro,13 the potential role of heterophilic cadherin interactions between neural crest cells and placode cell-derived neurons as they assemble the trigeminal ganglion has yet to be explored.\n\nTo address this question, we performed experiments to elucidate potential heterophilic interactions between Cadherin-7 and N-cadherin in the formation of the chick trigeminal ganglion. Our in vivo and in vitro biochemistry and imaging data indicate Cadherin-7 and N-cadherin physically interact during trigeminal ganglion assembly and that this involves heterophilic interactions between Cadherin-7, expressed in neural crest cells, and N-cadherin, found in placodal neurons. These findings further clarify the reciprocal relationship observed between coalescing neural crest cells and placodal neurons during trigeminal gangliogenesis, providing an additional molecular basis for this process.\n\n\nMethods\n\nFertilized chicken eggs (Gallus gallus) were obtained from the Department of Animal and Avian Sciences, University of Maryland, and Moyer’s Chicks, Inc. (PA), and incubated at 37°C in humidified incubators (EggCartons.com, Manchaug, MA, USA). Embryos were staged by the Hamburger-Hamilton (HH) staging method22 or by counting the number of somite pairs (somite stage, ss).\n\nNo ethical approval was required for this study for the chick embryos. At the stages of development being examined, the chick embryos used for our experiments are not considered live animals. According to the Office of Laboratory Animal Welfare (National Institutes of Health (NIH)), “the Public Health Service Policy on Human Care and Use of Laboratory Animals is applicable to proposed activities that involve live vertebrate animals. While embryonal stages of avian species develop vertebrae at a stage in their development prior to hatching, the NIH Office for Protection from Research Risks has interpreted “live vertebrate animal” to apply to avians (e.g., chick embryos) only after hatching.” Since our work does not utilize hatched chicks, no Institutional Animal Care and Use protocol for this work is necessary.\n\nFour different GRASP constructs were synthesized by GenScript (RRID:SCR_002891) to allow for incorporation of split GFP moieties (subunits 1-10 or subunit 11) into the extracellular domain of Cadherin-7 and N-cadherin, with the design based on similar plasmids generated in23 and available in Addgene (m-sGFP1-10::NLG1 (Addgene plasmid #44967; RRID:Addgene_44967) and m-sGFP11::NXN were gifts from Joshua Sanes (Addgene plasmid #44968; RRID:Addgene_44968)). Briefly, each plasmid from Addgene was modified to remove the respective insert (either NLG1 or NXN), and, in its place, we inserted the Cadherin-7 or N-cadherin cDNA sequence corresponding to the mature peptide. Sequence accuracy of constructs was confirmed by GenScript and expression of each cadherin was validated through immunocytochemistry.\n\nChinese hamster ovary (CHO) cells (ATCC Cat# CCL-61, RRID:CVCL_0214; American Type Culture Collection) were cultured in Ham’s F12 media (10-080, Corning/Cellgro) supplemented with 10% fetal bovine serum (Genesee Scientific Cat#25-514H). Mouse L cells (ATCC Cat# CRL-2648, RRID:CVCL_4536; American Type Culture Collection) were cultured in Dulbecco’s Modified Eagle Medium (DMEM; 11971-025, Gibco; Thermo Fisher Scientific, Inc.) supplemented with 10% fetal bovine serum. Transient transfection assays were carried out using the Lipofectamine 2000 reagent (Thermo Fisher Scientific, Inc., Cat#11668019). Cells were grown to 90% confluency, and transfections were performed according to the manufacturer’s instructions and according to the protocols outlined in.24,25 The chick N-cadherin-expressing (pCIG-N-cadherin) and empty (pCIG) vectors were gifts from Dr. Marianne Bronner (California Institute of Technology).\n\nTransfected cells grown in 10 cm plates or trigeminal ganglia were used for immunoprecipitations, with cells and embryonic tissue harvested as described previously by Refs. 14, 25, 26. Briefly, forming trigeminal ganglia were dissected, pooled, pelleted, flash-frozen in liquid nitrogen, and stored at -80°C. Cultured cells were scraped into 1X Phosphate-buffered Saline (1X PBS), pelleted, flash-frozen in liquid nitrogen, and stored at -80°C. Pellets were thawed on ice and lysed in lysis buffer (50 mM Tris pH 8.0, 150 mM NaCl, 1% IGEPAL CA-630) supplemented with cOmplete protease inhibitor cocktail tablets (Roche, Cat#04693124001) and 1 mM PMSF (Sigma Aldrich Cat#10837091001) for 30 minutes at 4°C with periodic mixing. Soluble fractions were collected following centrifugation at maximum speed for 15 minutes at 4°C (Microfuge 20R Centrifuge, Beckman Coulter, Inc., Cat#B31612), and protein concentration was quantified (BioPhotometer, Eppendorf, Cat#6131 26936) by Bradford assay (Thermo Fisher Scientific, Inc., Cat#1863028). Immunoprecipitations were carried out using protein A/G magnetic beads (Thermo Fisher Scientific, Inc., Cat#88802) according to the manufacturer’s instructions (Thermo Fisher Scientific, Inc.). Equivalent amounts of protein lysates (~120 μg) were incubated with 10 μg rabbit polyclonal N-cadherin antibody (Abcam Cat#ab12221, RRID:AB_298943) or normal rabbit IgG control (R&D Systems Cat#AB-105-C, RRID:AB_354266) overnight at 4°C with constant rotation. The following day, 0.25 mg washed protein A/G magnetic beads were incubated with the lysate/antibody mixture for one hour at room temperature with mixing. Following incubation, the samples were washed, equivalent volumes of SDS sample buffer were added, mixtures were boiled at 100°C for 10 minutes, magnetic beads were collected, and samples were loaded for immunoblotting as described below. Input amounts represent 5% (trigeminal ganglia) and 10% (cell culture) of the initial lysate amount used in the immunoprecipitation. Assays were conducted at least twice.\n\nImmunoblotting after immunoprecipitation was performed according to the protocol by Refs. 14, 25, 26. Samples were processed via SDS-PAGE (10% Mini-Protean TGX gel, BioRad #456-1034) in 1X Running Buffer (25 mM Tris (Thermo Fisher Scientific, Inc., Cat#BP-152-1), 192 mM glycine (Thermo Fisher Scientific, Inc., Cat#AC120070010), 0.1% sodium dodecyl sulfate (VWR, Cat#4095-02)) and then transferred to 0.45 μm BioTrace nitrocellulose membrane (Pall, Cat#66485) via wet transfer (Biorad, Mini-PROTEAN Tetra Vertical Cell for Mini Precast gels, Cat#1658004) in 1X Transfer Buffer (Running Buffer + 10% Methanol (Thermo Fisher Scientific, Inc., Cat#A452-4)) according to the manufacturer’s guidelines. For immunoblotting, membranes were blocked in blocking buffer (1X PBS + 0.1% Tween-20 (Sigma Aldrich, Cat#P1379-500ML)) (PTW) + 5% non-fat milk (Carnation Instant Nonfat Dry Milk). Next, primary antibodies against mouse monoclonal Cadherin-7 (1:150, DSHB, Cat#ccd7-1, RRID:AB_528111), rabbit polyclonal N-cadherin (1:1000, Abcam Cat#ab12221) or mouse monoclonal β-Actin (C4) (1:1000, Santa Cruz Biotechnology Cat#sc-47778, RRID:AB_626632) were diluted as indicated in blocking buffer and incubated overnight with shaking at 4°C. Unbound primary antibodies were washed off with PTW (three times, 10 minutes each), followed by incubation at room temperature for 45 minutes with the following secondary antibodies diluted in blocking buffer (1:10,000): goat anti-mouse polyclonal IgG (H&L) antibody peroxidase conjugated (Rockland Cat# 610-1302, RRID:AB_219656) or goat anti-rabbit polyclonal IgG (H&L) secondary antibody peroxidase conjugated (Rockland Cat# 611-1302, RRID:AB_219720). After washing three times, 10 minutes each, in PTW, proteins were detected using enhanced chemiluminescent substrates mixed in a 1:1 ratio (SuperSignal West Pico PLUS Chemiluminescent Substrate (Thermo Fisher Scientific, Inc., Cat#34580) or SuperSignal West Femto Maximum Sensitivity Substrate (Thermo Fisher Scientific, Inc., Cat#34095)). Immunoblot images for figures were gamma-modified and processed using Adobe Photoshop (RRID:SCR_014199) CC 2019 (20.0.6 release, Adobe Systems, San Jose, CA, USA).\n\nEmbryos collected at various stages, or cultured cells in two-well chamber slides (LAB-TEK, Cat#154461), were used for immunostaining. For the former, detection of various proteins was performed on 14 μm transverse sections following 4% paraformaldehyde (PFA) fixation overnight, gelatin embedding, and cryostat sectioning, according to the protocol described previously by.14,26–27 For the latter, cells were fixed in 4% PFA for 15 minutes, followed by immunocytochemistry. Tissue or cells were permeabilized by washing two times, 10 minutes each, in 1X PBS + 0.1% Triton X-100 (Sigma Aldrich, Cat#TX1568-1) (PBSTX), followed by a one hour blocking step of PBSTX + 10% sheep serum (Sigma Aldrich, Cat#S2263-100ML). All primary and secondary antibodies were diluted in 1X PBSTX + 5% sheep serum. The following antibodies and dilutions were used for immunostaining: mouse monoclonal anti-Cadherin-7 (1:50-1:70, DSHB Cat#ccd7-1); rat monoclonal anti-N-cadherin (1:50, DSHB Cat#MNCD2, RRID:AB_528119); mouse monoclonal anti-human natural killer-1 (HNK-1) (1:100, DSHB Cat#3H5, RRID:AB_2314644); and mouse monoclonal anti-Tubulin beta-3 chain (Tubb3) (1:500, Abcam Cat# ab78078, RRID:AB_2256751). The following secondary antibodies were used at 1:200-1:500 dilutions: goat anti-mouse polyclonal IgG (H + L) Cross-Adsorbed Secondary Antibody, Alexa Fluor 594 (Thermo Fisher Scientific Cat# A-11005, RRID:AB_2534073) and goat anti-mouse polyclonal IgG (H + L) Cross-Adsorbed Secondary Antibody, Alexa Fluor 647 (Thermo Fisher Scientific Cat# A-21235, RRID:AB_2535804) (for Cadherin-7); goat anti-rat polyclonal IgG (H + L) Cross-Adsorbed Secondary Antibody, Alexa Fluor 594 (Thermo Fisher Scientific Cat# A-11007, RRID:AB_10561522) (for N-cadherin); goat anti-mouse polyclonal IgM (Heavy Chain) Secondary Antibody (Thermo Fisher Scientific Cat# A-21238, RRID:AB_2535807) (for HNK-1); and goat anti-mouse polyclonal IgG2a Human ads-AF555 (SouthernBiotech Cat# 1080-32, RRID:AB_2794491) (for Tubb3). Sections were stained with 4′,6-diamidino-2-phenylindole (DAPI) to mark cell nuclei using DAPI-containing mounting media (DAPI Fluoromount-G, Southern Biotech, Cat#0100-20).\n\nFor sequential electroporation of both premigratory neural crest cells and trigeminal placode cells, unilateral chick neural tube electroporation to target neural crest cells contributing to the trigeminal ganglion was first performed, as described previously by.14,27 Briefly, GRASP constructs were introduced unilaterally into premigratory midbrain neural crest cells in developing 3 to 5 somite stage (3-5ss) chick embryos at a concentration of 2.0-2.5 μg/μl, using fine glass needles to fill the chick neural tube. Platinum electrodes were placed on either side of the embryo, and two 25 V, 25 ms electric pulses were applied across the embryo. Once embryos reached HH10-11 (10-13ss), a unilateral ectodermal electroporation was carried out (on the same side of the embryo that was electroporated previously) to target trigeminal placode cells.26 Electrodes were placed vertically on top of and below the embryo and three, 9 V pulses were delivered over 50 ms at 200 ms intervals. After electroporation, eggs were re-sealed with tape and parafilm and re-incubated for the desired time period (approximately 36 hours to reach HH15-16) prior to harvesting for fixation and transverse sectioning, which was carried out according to the protocol by Ref. 14.\n\nFor all experiments, images of at least five serial transverse sections through a minimum of five embryos were acquired with the LSM Zeiss 800 confocal microscope with Airyscan detection (Carl Zeiss Microscopy, Thornwood, NY, USA) at 20X magnification. Laser power, gain, and offset were kept consistent for the different channels during all experiments where possible. ZEN Digital Imaging for Light Microscopy (RRID:SCR_013672), version 2.3 software (Carl Zeiss Microscopy) and Adobe Photoshop CC 2019 (20.0.6 release) were used for image processing. Equivalent functions for image processing can be performed on Fiji (RRID:SCR_002285), which is freely available.\n\n\nResults\n\nCranial neural crest cells and trigeminal placodal neurons express distinct cadherins, Cadherin-7 and N-cadherin, respectively, during early trigeminal gangliogenesis.14,16 Given these findings, we sought to determine whether these specific cadherins facilitated trigeminal ganglion assembly through heterophilic interactions. To address this, we first evaluated Cadherin-7 and N-cadherin antibody specificity by performing co-immunoprecipitation assays in L cells lacking endogenous cadherins28 that were transfected to express chick N-cadherin, and in the forming trigeminal ganglia of HH15-16 chick embryos (Figure 1).47\n\nLysate from L cells and HH15-16 trigeminal ganglion tissue was incubated with either an antibody against N-cadherin or with whole rabbit IgG serum as a control. Immunoprecipitated proteins were captured with protein A/G beads, separated by SDS-PAGE, followed by immunoblotting for N-cadherin (A) and Cadherin-7 (B). Lanes 1-10 are as follows: 1) Input, lysate from pCIG empty vector-transfected L cells (L-control); 2) L-control lysate following IP with rabbit IgG; 3) L-control L lysate after IP with N-cadherin antibody; 4) Input, lysate from L cells transfected with pCIG-N-cadherin (L-N-cad); 5) L-N-cad lysate following IP with rabbit IgG; 6) L-N-cad lysate following IP with N-cadherin antibody; 7) Protein ladder; 8) Input, trigeminal ganglia lysate; 9) trigeminal ganglia lysate following IP with rabbit IgG; and 10) trigeminal ganglia lysate following IP with N-cadherin antibody. Arrowheads point to N-cadherin (A) or Cadherin-7 (B), respectively, while asterisks identify Cadherin-7 immunoreactive products as observed previously.14 N-cadherin, neural cadherin; IP, immunoprecipitation.\n\nAnalysis of immunoprecipitated proteins by SDS-PAGE followed by immunoblotting for N-cadherin revealed that, as expected in control L cells, N-cadherin is not expressed (Figure 1A, lane 1), nor is it noted in the control IgG or N-cadherin antibody immunoprecipitations (Figure 1A, lanes 2 and 3). Transient transfection of L cells with pCIG-N-cadherin, however, led to the presence of N-cadherin, in both input (Figure 1A, lane 4; arrowhead points to N-cadherin) and after N-cadherin immunoprecipitation (Figure 1A, lane 6), but not with the rabbit IgG serum (Figure 1A, lane 5). Notably, N-cadherin within the forming trigeminal ganglia (Figure 1A, lane 8) was also detected in N-cadherin immunoprecipitates with this antibody (Figure 1A, lane 10), but not with the rabbit IgG serum (Figure 1A, lane 9). These data indicate that the N-cadherin antibody can effectively immunoprecipitate N-cadherin from both N-cadherin-transfected L cells and trigeminal ganglia tissue, providing us with a key experimental tool to identify other proteins that physically interact with N-cadherin in vivo.\n\nTo this end, we next performed immunoblotting using a validated Cadherin-7 antibody13,14 (Figure 1B). Our data again reveal antibody specificity, with no bands appearing in the cell culture input (Figure 1B, lane 1) or in vector- or N-cadherin-transfected cells after immunoprecipitation (Figure 1B, lanes 2-6). A band corresponding to Cadherin-7 is observed in the trigeminal ganglia lysate input sample (Figure 1B, lane 8, arrowhead), along with immunoreactive lower molecular weight bands (Figure 1B, asterisk, *) containing portion(s) of the Cadherin-7 extracellular domain, as observed in our prior work.14 Strikingly, we also observed Cadherin-7 after N-cadherin pull-down (Figure 1B, lane 10, arrowhead), but not with the control IgG serum (Figure 1B, lane 9). These findings reveal N-cadherin and Cadherin-7 physically interact in vivo. As N-cadherin is noted in trigeminal placodal neurons and cranial mesenchyme16 but only neural crest cells express Cadherin-7,14 our data suggest heterophilic interactions between Cadherin-7 in neural crest cells and N-cadherin in placodal neurons and/or the mesenchyme.\n\nGiven the results of our pull-down experiments, we hypothesized that physical interactions between Cadherin-7 in neural crest cells and N-cadherin in placodal neurons mediate, in part, the successful aggregation of these cell types during trigeminal gangliogenesis. To address this, we adapted and modified a GRASP assay to evaluate interactions specifically between these two cadherins, both in vitro and in vivo. GRASP relies upon functional complementation (i.e., GFP fluorescence) between two non-fluorescing or split GFP fragments (GFP1-10, GFP11). Reconstitution of GFP can only occur when the split GFP molecules are in close proximity to each other, as observed in other systems that defined interactions between extracellular domains of two membrane proteins.23,29–31 We designed Cadherin-7 and N-cadherin GRASP vectors (Figure 2) with GFP subunits fused in frame to the respective cadherin extracellular domain (Cadherin-7 GFP1-10, Cadherin-7 GFP11, N-cadherin GFP1-10, N-cadherin GFP11; GenScript). Constructs were based on GRASP plasmids developed by the Sanes lab (Addgene), which generate intact GFP fluorescence/puncta due to neuroligin-neurexin interactions, with no GFP noted with single constructs.23\n\nCartoon diagram showing GFP-cadherin fusion proteins that were constructed by joining the kappa light chain to distinct GFP subunits (1–10, or 11), followed by a linker region and then the mature cadherin peptide. GFP, green fluorescent protein; GRASP, GFP reconstitution across synaptic partners; N-cad, neural cadherin; Cad7, Cadherin-7.\n\nWe first showed that all constructs expressed their respective cadherins by transfecting CHO cells, which lack endogenous cadherins,32 with each GRASP construct, followed by immunostaining for each cadherin (Figure 3A’, B’, C’, D’, arrows). Importantly, no GFP fluorescence was noted under any condition, as expected. We next evaluated the specificity of the split GFP moieties to generate GFP by co-transfecting CHO cells with the same split GFP constructs, but fused to a different cadherin (i.e., Cadherin-7 GFP1-10 and N-cadherin GFP1-10). In these control experiments, expression of each cadherin was observed once again (Figure 4A”’, B”’, arrows), but no GFP was reconstituted, reinforcing the specificity of the assay.\n\nSingle transfections of Cad7 GFP1-10 (A-A’), Cad7 GFP11 (B-B’), N-cad GFP1-10 (C-C’), and N-cad GFP11 (D-D’) were conducted in CHO cells, followed by immunocytochemistry for Cad7 (A’, B’, red) or N-cad (C’, D’, red). GFP fluorescence was also examined in the appropriate microscope channel (488) but not observed. Arrows point to cadherin expression in transfected cells. DAPI (blue), cell nuclei. Scale bar in (A) is 50 μm and applies to all images. GFP, green fluorescent protein; GRASP, GFP reconstitution across synaptic partners; CHO, Chinese hamster ovary; N-cad, neural cadherin; Cad7, Cadherin-7.\n\nCo-transfection of CHO cells with Cad7 GFP1-10 + N-cad GFP1-10 (A-A”’), or Cad7 GFP11 + N-cad GFP11 (B-B”’), was performed, followed by immunocytochemistry for Cad7 (A’, A”’, B’, B”’, purple) and N-cad (A”, A”’, B”, B”’, red). GFP fluorescence was also examined in the appropriate microscope channel (488) but not observed. Arrows point to cadherin expression in co-transfected cells. DAPI (blue), cell nuclei. Scale bar in (A) is 50 μm and applies to all images. GFP, green fluorescent protein; GRASP, GFP reconstitution across synaptic partners; CHO, Chinese hamster ovary; N-cad, neural cadherin; Cad7, Cadherin-7.\n\nNext, we addressed whether cis interactions between complementary split GFP constructs could generate an intact GFP molecule in vitro. To this end, we co-transfected CHO cells with complementary split GFP constructs expressing the same cadherin (Figure 5) and examined cadherin expression by immunostaining, as well as checked for GFP fluorescence. GFP expression/puncta was detected with both Cadherin-7- (Figure 5A, A”, arrows) or N-cadherin- (Figure 5B, B”, arrows) expressing split GFP constructs, along with expression of each respective cadherin (Figure 5A’, B’), demonstrating effective GFP reconstitution via homophilic cadherin interactions.\n\nCHO cells were co-transfected with complementary split GFP constructs expressing the same cadherin (Cad7 GFP1-10 and Cad7 GFP11 (A-A”); N-cad GFP1-10 and N-cad GFP11, (B-B”)), followed by immunocytochemistry for Cad7 (A’, A”, purple) or N-cad (B’, B”, red). GFP fluorescence was also examined in the appropriate microscope channel (488, A, A”, B, B”, green). Arrows point to GFP fluorescence in transfected cells, indicative of physical interactions between each split GFP-expressing cadherin. DAPI (blue), cell nuclei. Scale bar in (A) is 50 μm and applies to all images. GFP, green fluorescent protein; GRASP, GFP reconstitution across synaptic partners; CHO, Chinese hamster ovary; N-cad, neural cadherin; Cad7, Cadherin-7.\n\nTo evaluate this in the context of the potential formation of heterophilic cadherin complexes, the same co-transfection experiment was conducted in CHO cells but this time using complementary split GFP constructs fused to a different cadherin (Figure 6). Our results revealed GFP reconstitution (Figure 6A, A”’, B, B”’, arrows) and cadherin expression (Figure 6A-A”’, B-B”’), pointing to the ability of Cadherin-7 and N-cadherin to interact in cis and form heterophilic complexes, further validating our in vivo biochemistry results in the chick trigeminal ganglion.\n\nCad7 GFP1-10 and N-cad GFP11 (A-A”’), or N-cad GFP1-10 and Cad7 GFP11 (B-B”’), were co-transfected into CHO cells, followed by immunocytochemistry for Cad7 (A’, A”’, B’, B”’, purple) and N-cad (A”, A”’, B”, B”’, red). GFP fluorescence was also examined in the appropriate microscope channel (488, A, A”’, B, B”’, green). Arrows point to GFP fluorescence in transfected cells, indicative of physical interactions between each split GFP-expressing cadherin. DAPI (blue), cell nuclei. Scale bar in (A) is 50 μm and applies to all images. GFP, green fluorescent protein; GRASP, GFP reconstitution across synaptic partners; CHO, Chinese hamster ovary; N-cad, neural cadherin; Cad7, Cadherin-7.\n\nTo corroborate our findings and examine cadherin intercellular interactions during trigeminal ganglion assembly in vivo, we turned to a sequential electroporation assay in which a Cadherin-7 split GFP construct was first electroporated into premigratory neural crest cells, followed by a second electroporation of a complementary N-cadherin split GFP construct to target trigeminal placode cells in the surface ectoderm (Figure 7). Transverse sections taken from electroporated embryos were processed for immunohistochemistry to identify neural crest cells and placodal neurons within the forming trigeminal ganglion. Remarkably, we observed puncta of GFP expression between neural crest cells (labeled by HNK-1; Figure 7A, A”, A”’, B, B”, B”’) and placodal neurons (labeled by Tubb3; Figure 7A, A’-A”’, B’-B”’) in the presence of the appropriate split GFP constructs (Figure 7A”’, B”’, arrows). These data indicate Cadherin-7 and N-cadherin are in close proximity to interact in trans and permit the reconstitution of GFP in vivo, even in different cell types. Together with our biochemistry data and results in cultured cells, our findings support the assertion that heterophilic interactions between Cadherin-7 in neural crest cells and N-cadherin in placodal neurons occur during trigeminal gangliogenesis.\n\nSequential electroporation in the chick were conducted as follows: Premigratory NCCs were first electroporated with Cad7 GFP1-10 (A-A”’) or Cad7 GFP11 (B-B”’), followed by electroporation of trigeminal PCs with N-cad GFP11 (A-A”’) or N-cad GFP1-10 (B-B”’), respectively. Immunohistochemistry for HNK-1 (purple, marks neural crest cells; A, A”, A”’, B, B”, B”’) and Tubb3 (red, marks neurons which are all placode-derived at this stage; A’-A”’, B’-B”’) was performed. GFP signal/puncta (A”’, B”’, green, arrows) was captured in the appropriate channel (488). (A”’) and (B”’) are higher magnification images of the boxed region in (A”) and (B”), respectively. DAPI (blue), cell nuclei. Scale bar in (A) is 75 μm and applies to all images but is 25 μm for (A”’) and (B”’). N-cad, neural cadherin; Cad7, Cadherin-7; NCCs, neural crest cells; PCs, placode cells; GFP, green fluorescent protein; HNK-1, human natural killer-1; Tubb3, Tubulin beta-3 chain.\n\n\nDiscussion\n\nCranial neural crest cells and placode cells initially form in close proximity but become spatially separated as development ensues.8,33–35 While these cells give rise to distinct derivatives, they will both form sensory neurons of the trigeminal ganglion, innervating much of the head and face to relay information related to pain, touch, and temperature to the central nervous system.3–5 The cellular origin of the trigeminal ganglion has been known for decades7,10,36; however, molecular mechanisms mediating early interactions between neural crest cells and placodal neurons to build the trigeminal ganglion have not been well characterized. In the chick embryo, studies uncovered the importance of cadherin-mediated interactions, as distinct cadherins are expressed by neural crest cells (Cadherin-7)13,14 and placode cells and their neuronal derivatives (N-cadherin).16 The presence of two different cadherins on these coalescing cells begs the question as to whether heterophilic interactions exist between them to allow for proper trigeminal ganglion formation, particularly since cells expressing these cadherins can form mixed aggregates in vitro.13\n\nOur studies now address this question through the use of biochemistry and an adapted GRASP assay to examine cadherin interactions during trigeminal ganglion development. Through in vitro transfection experiments and use of embryonic trigeminal ganglia tissue, we demonstrate a physical interaction between Cadherin-7 and N-cadherin. This is the first report to reveal, biochemically, that Cadherin-7-N-cadherin complexes can form and, notably, are present while the trigeminal ganglion assembles. While we cannot rule out the presence of other protein(s) in the embryo to serve as a “bridge” to allow these cadherins to associate, these data still provide strong evidence that these interactions do exist in vivo.\n\nTo generate the trigeminal ganglion, Cadherin-7-expressing cranial neural crest cells first migrate through the embryonic mesenchyme to the trigeminal ganglionic anlage. Here, they intermingle with newly differentiated, N-cadherin-expressing trigeminal placode-derived neurons, which have delaminated from the surface ectoderm and have also migrated through the mesenchyme. Since the cranial mesenchyme expresses N-cadherin, it is possible that the Cadherin-7-N-cadherin complexes we detected through our biochemistry studies represent interactions between Cadherin-7 on neural crest cells and N-cadherin expressed in mesenchymal cells. However, based upon the abundance of neurons in relation to the mesenchyme in dissected trigeminal ganglia, we think the primary source of N-cadherin in these interactions comes from the placodal neurons. Moreover, prior work revealed that neural crest cells form corridors through which placodal neurons migrate, thereby providing a more permissive migratory environment compared to the cranial mesenchyme.37,38 As such, neural crest cells and placodal neurons are tightly juxtaposed during the assembly of the trigeminal ganglion, making it more likely that the interactions we are detecting arise from Cadherin-7 on neural crest cells and N-cadherin on placodal neurons.\n\nTo further define and directly visualize these heterophilic cadherin interactions, we conducted a GRASP assay in cell culture and in the embryo. We generated two split GFP constructs (GFP domains 1-10 or GFP domain 11) fused to both Cadherin-7 and N-cadherin and examined the ability of these cadherins to associate in cis and in trans to generate GFP. Through cell culture co-transfection experiments, we demonstrated that GFP could be reconstituted as long as the split GFP constructs were complementary, providing further evidence that Cadherin-7 and N-cadherin can interact in cis. Importantly, no GFP was generated after co-transfection of like split GFP moieties fused to different cadherins, pointing to the specificity of the GFP reconstitution.\n\nWith these tools, we next explored the ability of Cadherin-7-expressing neural crest cells to associate with N-cadherin-expressing placodal neurons. Sequential electroporation experiments were conducted in which complementary split GFP constructs were introduced into neural crest cells (Cadherin-7 split GFP construct) followed by placode cells (N-cadherin split GFP construct). Because of the anatomy of the chick embryo at the time of electroporation and tissue of origin of neural crest cells (dorsal neural folds) and placode cells (surface ectoderm), we can precisely, and independently, target each cell type. Notably, we observed GFP puncta at sites where neural crest cells and placodal neurons come into contact, visualized on sections taken through the developing trigeminal ganglion. These data reveal that Cadherin-7 and N-cadherin can interact in trans in different cell populations, providing insight into the ability of different cadherin-expressing cells to associate in vivo. Although the number of GFP puncta was not extraordinarily high, this is to be expected given the nature of the electroporation, in which only a small amount of each split GFP construct was electroporated into each cell type in order to avoid potential artifacts of overexpression.\n\nOther pathways have been discovered to regulate cellular interactions occurring during initial chick trigeminal ganglion coalescence, including Slit1-Robo2,11,16 Wnt,39,40 Neuropilin/Semaphorin,41,42 and various growth factors43 (e.g., Platelet-Derived Growth Factor44), with many of these also identified in the developing mouse trigeminal ganglion.45,46 In chick embryos, Robo2 signaling likely modulates levels of N-cadherin post-translationally, but the mechanisms underlying this are still not well characterized. Upstream pathways regulating Cadherin-7 expression in neural crest cells also remain obscure, but it is plausible that the preceding signal transduction pathways could impact the expression of Cadherin-7 and/or N-cadherin during trigeminal gangliogenesis. Future studies aimed at addressing this question will provide important insights into the regulation of neural crest-placodal neuron migration and adhesion.\n\nThe juxtaposition of Cadherin-7-expressing neural crest cells and N-cadherin-expressing placodal neurons in the forming trigeminal ganglion hinted at the possibility that heterophilic interactions between these two cadherins could, in part, mediate this process. While the functional roles of each cadherin in trigeminal ganglion assembly have been well described, less attention was paid to the importance of their expression in building the ganglion. Cultured cells expressing either Cadherin-7 or N-cadherin can form intermingled aggregates, supporting the notion of heterophilic interactions, but it was not evaluated in vivo until our studies. We now provide data uncovering a physical interaction between Cadherin-7 in neural crest cells and N-cadherin in placodal neurons within the trigeminal ganglion. Altogether, these findings shed light on the molecular mechanisms underscoring intercellular interactions requisite for trigeminal ganglion assembly during early chick embryonic development.\n\n\nData availability\n\nDigital Repository at the University of Maryland, Animal & Avian Sciences Research Works: Neural crest cell-placodal neuron interactions are mediated by Cadherin-7 and N-cadherin during early chick trigeminal ganglion assembly. https://doi.org/10.13016/llyh-dppy.47\n\nThis project contains the following underlying data:\n\n‐ Figure 1: Raw western blot data (Original raw tiff files for the immunoblotting experiments)\n\n‐ Figure 2: Plasmids.pdf (GRASP cadherin plasmid sequences)\n\n‐ Figure 3: Transfection images for single split GFP cadherin constructs\n\n‐ Figure 4: Transfection images for double, non-complementary, split GFP constructs with different cadherins\n\n‐ Figure 5: Transfection images for double, complementary, split GFP constructs with the same cadherin\n\n‐ Figure 6: Transfection images for double, complementary, split GFP constructs with different cadherins\n\n‐ Figure 7: Tissue section images following electroporation of complementary split GFP constructs into neural crest cells and placode cells\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).",
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PubMed Abstract\n\nFreter S, Fleenor SJ, Freter R, et al.: Cranial neural crest cells form corridors prefiguring sensory neuroblast migration. Development. 2013; 140(17): 3595–3600. PubMed Abstract | Publisher Full Text\n\nSandell LL, Butler Tjaden NE, Barlow AJ, et al.: Cochleovestibular nerve development is integrated with migratory neural crest cells. Dev. Biol. 2014; 385(2): 200–210. PubMed Abstract | Publisher Full Text\n\nShiau CE, Hu N, Bronner-Fraser M: Altering Glypican-1 levels modulates canonical Wnt signaling during trigeminal placode development. Dev. Biol. 2010; 348(1): 107–118. PubMed Abstract | Publisher Full Text\n\nShigetani Y, Howard S, Guidato S, et al.: Wise promotes coalescence of cells of neural crest and placode origins in the trigeminal region during head development. Dev. Biol. 2008; 319(2): 346–358. PubMed Abstract | Publisher Full Text\n\nGammill LS, Gonzalez C, Bronner-Fraser M: Neuropilin 2/semaphorin 3F signaling is essential for cranial neural crest migration and trigeminal ganglion condensation. Dev. Neurobiol. 2007; 67(1): 47–56. PubMed Abstract\n\nSchwarz Q, Vieira JM, Howard B, et al.: Neuropilin 1 and 2 control cranial gangliogenesis and axon guidance through neural crest cells. Development. 2008; 135(9): 1605–1613. PubMed Abstract | Publisher Full Text\n\nMcCabe KL, Shiau CE, Bronner-Fraser M: Identification of candidate secreted factors involved in trigeminal placode induction. Dev. Dyn. 2007; 236(10): 2925–2935. PubMed Abstract | Publisher Full Text\n\nMcCabe KL, Bronner-Fraser M: Essential role for PDGF signaling in ophthalmic trigeminal placode induction. Development. 2008; 135(10): 1863–1874. PubMed Abstract | Publisher Full Text\n\nMaynard TM, Horvath A, Bernot JP, et al.: Transcriptional dysregulation in developing trigeminal sensory neurons in the LgDel mouse model of DiGeorge 22q11.2 deletion syndrome. Hum. Mol. Genet. 2020; 29(9): 1580. PubMed Abstract | Publisher Full Text\n\nKarpinski BA, Maynard TM, Bryan CA, et al.: Selective disruption of trigeminal sensory neurogenesis and differentiation in a mouse model of 22q11.2 deletion syndrome. Dis. Model. Mech. 2022; 15(2). PubMed Abstract | Publisher Full Text\n\nHalmi C, Wu C-Y, Taneyhill L: Neural crest cell-placodal neuron interactions are mediated by Cadherin-7 and N-cadherin during early chick trigeminal ganglion assembly. [Dataset]. Digital Repository at the University of Maryland. Animal & Avian Sciences Research Works. 2022. Publisher Full Text"
}
|
[
{
"id": "143220",
"date": "18 Jul 2022",
"name": "Kristin Bruk Artinger",
"expertise": [
"Reviewer Expertise Neural crest and craniofacial developoment"
],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe manuscript by Halmi et al examines interactions between Cad-7 and N-cadherin during assembly of the trigeminal ganglia. Using biochemistry and chick overexpression models, and novel GRASP imaging, the authors that Cadherin-7 and N-cadherin interact to build the ganglia. This paper demonstrates a novel interaction in vitro and in vivo. It is well written, shows beautiful imaging, and would be of general interest to readers who are interested in cadherins and ganglia assembly. There are some areas of clarification that would strengthen the manuscript. My comments are below:\nFurther clarification to the cell types used is warranted here. It seems the western is done in L cells while staining in Cho cells. Is it because they are not expressed? Is there a way to quantify the levels as compared to endogenous levels in cells that express them?\n\nIt seems that in, in vitro that the Cad7-Cad7 and N-cad-N-cad interaction is much stronger than the Cad-7-N-cad interaction and maybe not as broadly expressed? Here also in Figure 6, the morphology of the cells looks different than in Figure 5. Is this true? Please add this to the results.\n\nIn vivo, the gfp is localized in very few cells. What does the Cad7-Cad7 and N-cad-N-cad interaction look like in the trigeminal ganglia? Is it similarly low or higher? This would add to the data presented here. It also makes me wonder if the interaction changes at different developmental times.\n\nIn general, quantification of the number of fluorescent puncta would be useful for all the studies but in particular in vivo. And does the intensity correlate with the strength of the interaction? If so, these would also be interesting to quantify.\n\nIs the work 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": "9076",
"date": "12 Dec 2022",
"name": "Lisa Taneyhill",
"role": "Author Response",
"response": "We thank Dr. Artinger for their thoughtful comments on the manuscript. We have responded to each point below and will make necessary updates to the manuscript, where applicable. We apologize for the lack of clarity in the use of two cell lines to examine cadherin expression. Both L cells (from mouse) and CHO cells lack endogenous cadherins (Hong et al., 2010). These cell lines were chosen to allow for ectopic expression of chick cadherins and to prevent any “interference” in the GRASP assay with any other cadherins that might be expressed in them. We initially overexpressed chick N-cadherin in L cells to test and ultimately demonstrate N-cad antibody specificity for immunoprecipitation (old Figure 1), as this experiment takes far less time to perform versus testing antibodies on embryonic tissue. However, we realize that the inclusion of this L cell line data in (old) Figure 1 has served as a source of confusion. To alleviate this, we have now modified the blots in Figure 1 to only include the in vivo data from the trigeminal ganglia lysate. The input sample in each of these blots, along with our prior publication (Wu and Taneyhill, 2019), reveals that both the N-cadherin and Cadherin-7 antibodies work by immunoblotting for their respective antigens. Moreover, the IgG control and N-cadherin antibody immunoprecipitation lanes indicate that the N-cadherin antibody is able to immunoprecipitate endogenous N-cadherin in trigeminal ganglia lysates. Given this, we do not think it is necessary to quantify N-cadherin and/or Cadherin-7 expression in other cell lines as our main point was to simply use the L cell transfection system to validate our antibodies. We are sorry again for the confusion this has caused and hope the Reviewer finds this solution acceptable. We thank Dr. Artinger for sharing their insights on the cadherin interactions observed in our GRASP cell culture assays. We agree that the homophilic cadherin interactions appear to be much stronger than the heterophilic cadherin interactions in the in vitro GRASP assay, and this is also apparent after quantifying the number of GFP-positive cells (new Table 1). These findings are in agreement with what has been published previously on dissociation constant measurements for homophilic versus heterophilic cadherin interactions in cell aggregates in vitro (Katsamba et al., 2009). Moreover, N-cadherin homophilic interactions confer a much higher degree of adhesivity than those mediated by Cadherin-7 (Chu et al., 2006). After careful review of our GRASP data, we also agree with Dr. Artinger’s assessment regarding the morphology of the cells. Generally, transfection of cells with like cadherins and complementary GRASP constructs gives rise to cells that exhibit a round shape or are only somewhat fibroblastic, with few protrusions emanating from the cell (Figure 5). Conversely, transfection of cells with different cadherins and complementary GRASP constructs causes cells to adopt a much more fibroblastic, and often spindly, morphology (Figure 6). This could be due to the presence of both a Type I (N-cadherin) and Type II (Cadherin-7) cadherin in these cells. A parallel to this can be found in vivo with respect to the overlapping expression domains of N-cadherin and Cadherin-7 in the developing chick spinal cord. These neuroepithelial cells are organized in a pseudostratified manner and thus exhibit a spindly morphology as they are densely packed within the neural tube/forming spinal cord (Lin et al., 2014). We have now added this text to the Results section and thank Dr. Artinger for sharing these observations, as they have strengthened the manuscript. Dr. Artinger raises an intriguing point about the Cadherin-7-Cadherin-7 and N-cadherin-N-cadherin interactions in vivo and how they compare to the GFP puncta (representative of Cadherin-7-N-cadherin interactions) we observe. Unfortunately, we are unable to make exact correlations between cadherin expression and GFP puncta because the presence of GFP relies upon each cell expressing its respective complementary cadherin split GFP construct, but the electroporation itself is mosaic. As such, not every neural crest cell or placode cell will be expressing a cadherin split GFP construct. However, we have now added a new figure (Figure 10) to the manuscript showing endogenous cadherin expression in neural crest cells (Cadherin-7) and placode cells (N-cadherin) in the forming trigeminal ganglion. As evident by these images, Cadherin-7-expressing neural crest cells are positioned next to N-cadherin-expressing placode cells. The majority of interactions at this stage, though, appear between adjacent neural crest cells or placode cells (i.e., cadherin homophilic interactions). These findings are in keeping with the GRASP assay data shown in (new) Figures 7 and 8 and the quantification we have performed (Table 2, see below). We thank Dr. Artinger for this useful suggestion about puncta quantification. We have examined a minimum of four serial sections taken through the forming trigeminal ganglion in at least six embryos after sequential electroporation of respective cadherin split GFP constructs into neural crest cells and then placode cells. Through these analyses, we have now quantified the number of GFP-positive regions observed and report these data in the manuscript. We note a statistically significant increase in the number of GFP-positive regions in the N-cad GFP11 + Cad7 GFP1-10 experiment vs. the N-cad GFP1-10 + Cad7 GFP11 experiment (p = 0.014). Interestingly, we observed similar results in our in vitro co-transfection experiments. While we might expect these numbers to be similar, the difference could be explained by the ability of cells generally to effectively express N-cad GFP1-10, or, conversely, the ability of cells to express Cad7 GFP11. We favor the former idea, however, for the following reasons. Given that the N-cadherin coding sequence is larger than the Cadherin-7 coding sequence (~300 nucleotides difference), coupled with the greater size of the split GFP1-10 moiety, it is possible that cells do not readily express N-cad GFP1-10 (compared to N-cad GFP11). Alternatively, perhaps there are inherent differences in the ability of neural crest cells and placode cells to transcribe and translate expression constructs like these, with neural crest cells more easily expressing Cad7 GFP1-10 compared to placode cells expressing N-cad GFP1-10. Finally, the heterophilic interactions we are examining occur in trans. As such, they are dependent upon a cadherin split GFP construct getting not only electroporated but also appropriately trafficked, and correctly targeted, to a region of the plasma membrane, where it will then be in close proximity to a complementary split cadherin GFP construct on the other cell type. For these reasons, fewer heterophilic interactions may ensue in a given electroporated tissue."
}
]
},
{
"id": "147854",
"date": "13 Sep 2022",
"name": "Paolo E. Forni",
"expertise": [
"Reviewer Expertise Developmental neurobiology"
],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 report by Caroline A. Halmi and coworkers explored the potential formation of Cadherin-7-N-cadherin complexes during trigeminal ganglion assembly. The authors suggested that the neural crest-derived cells bind to the placodal-derived neurons via heterophilic Cadherin-7-N-cadherin binding. To test this hypothesis the authors adopted classic pull-down experiments and an elegant GRASP assays approach. The latter was used both in cell culture and in the embryos. The GRASP assay was designed in order to express complementary split GFP constructs into neural crest cells (Cadherin-7 split GFP construct) and placode cells (N-cadherin split GFP construct). In this type of assay, GFP can only be detected when the split fusion proteins directly interact. The data presented are compelling and the conclusions of the paper are relevant and of broad interest to the community.\nI suggest some changes that would improve the quality of the manuscript:\nAll the figures would benefit from some quantifications and statistics supporting the data shown in the images. Moreover, in the legends, the authors should indicate the number of replicates.\n\nFig.7 The quality of the pictures is not impressive. The combination of colors Red/magenta/green in A’’ A’’’ and B’’ B’’’ is very hard to read. The author should try different color combinations. The antigen names on the figures are hard to see, consider using a bold font and outline. Moreover, it would be good to have a low-mag image and diagram illustrating where the image was taken in context to the developing chick head. This would help the readers to understand the morphology of the region being described.\n\nIs the work 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": "9077",
"date": "12 Dec 2022",
"name": "Lisa Taneyhill",
"role": "Author Response",
"response": "We thank Dr. Forni for their insightful comments on the manuscript. We have responded to each point below and will make necessary updates to the manuscript, where applicable. We apologize for the lack of quantification and statistics in our results, a point raised by Dr. Artinger and Dr. Wray. To this end, we have now quantified GFP-positive regions in our in vivo experiments and report a statistically significant increase in the number of GFP-positive regions in the Cad7 GFP1-10 + N-cad GFP11 electroporated embryos vs. the Cad7 GFP11 + N-cad GFP1-10 electroporated embryos (p = 0.014). We discuss possible reasons for this in the Discussion section, speculating that the reduced number of GFP-positive puncta in the latter could be due to the general larger size of the N-cad GFP1-10 plasmid, potentially precluding robust expression of this construct, and/or intrinsic differences in the ability of neural crest and placode cells to transcribe and translate expression constructs. Although it was not possible to distinguish individual GFP-positive puncta in the cell culture experiments, we have reported the percentage of GFP-positive cells in this assay, along with statistical comparisons. While there is no statistically significant difference in the number of GFP-positive cells after transfection of homophilic split GFP constructs (i.e., Cad7 GFP1-10 + Cad7 GFP11 or N-cad GFP1-10 + N-cad GFP11), we note a statistically significant difference in the number of GFP-positive cells when comparing Cad7 GFP1-10 + N-cad GFP11-transfected cells to Cad7 GFP11 + N-cad GFP1-10-transfected cells, with increased GFP noted in the former (p = 0.013). These findings are in keeping with our in vivo results and have been incorporated into the revised manuscript. For each figure, we have also included the number of replicates in the figure legends. We appreciate the feedback on Figure 7 from Dr. Forni and have modified this figure (and the other in vivo figures) based on his suggestions. These include bolding the antigen labels and placing them on a different color background, changing the antigen colors in the high magnification images to better visualize different cell populations, adding a lower magnification image of the immunohistochemistry, and including a cartoon diagram to illustrate the location of the image in the context of the developing head."
}
]
},
{
"id": "148069",
"date": "14 Sep 2022",
"name": "Susan Wray",
"expertise": [
"Reviewer Expertise GnRH neuroendocrine cells and olfactory placode development"
],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis paper examines interactions between Cad-7 (expressed in neural crest cells) and N-cadherin (expressed in placodal cells) during formation of the trigeminal ganglia. Previous work from this lab showed that depletion of Cad-7 in neural crest cells alters placodal cell shape/orientation while overexpression changed trigeminal structure. The current study examines whether heterotypic interactions between these two cadherins occur. Using IP biochemical methods on trigeminal ganglion tissue, the authors show that Cad-7 can interact with N-cadherin. In addition, experiments using ‘split GFP’ fragments in the developing chick trigeminal ganglion are provided that support the IP results. Together the data strengthen the argument that heterotypic interactions are involved in trigeminal gangliogenesis.\nClarifications/revisions that would strengthen the manuscript.\n\nFigure 6. It appears that the N-cad GFP1-10 + Cad7 GFP11 had more robust GFP puncta than the reverse group. Was this noticed in the trigeminal experiments? This brings up the issue of GFP quantification. Some measurements in both the cell lines as well as in vivo would be informative.\n\nExpression of GFP in ganglia electroporated with GRASP constructs. This is a very interesting experiment. However, it is difficult to understand all that is going on in Fig. 7. First, the methods state that the confocal imaging was performed at 20X magnification. Thus, I believe the images in Figure 7A”’ and B’’’ are enlargements of the images in A” and B”. To resolve where the GFP puncta occur, 60X or 100X may be required, as well as a few non-stacked single Z-plans. This would allow the reader to see where the combination is occurring. Second, the number of GFP expressing puncta is very low. This is dealt with in the discussion by saying “Although the number of GFP puncta was not extraordinarily high, this is to be expected given the nature of the electroporation, in which only a small amount of each split GFP construct was electroporated into each cell type in order to avoid potential artifacts of overexpression.” However, a control experiment to aid the reader would be an additional group that receives PCN-cad GFP1-10 + PCN-cad GFP11 at the same ‘concentration’ as electroporated for the two mixed GRASPs. Minor changes to improve this figure would be to place all labels on white or black inset, since difficult to see staining labels in most panels and in A and B”’ to include DAPI to show cell bodies which would aid the reader to understand where GFP is being detected.\n\nBelow are some minor issues/suggestions.\nIntroduction –\nThe trigeminal ganglion, the largest of the cranial ganglia, contains three sensory branches…. is the clause necessary for the reader to know? Could be deleted making read through easier.\n\nrevision suggested - More recent studies of confirmed Cadherin-7 protein in chick migratory cranial neural crest cells contributing to the trigeminal ganglion.14 These findings further clarify the reciprocal relationship observed\n\nEthical Approval - I believe only these two sentences are needed\nNo ethical approval was required for this study for the chick embryos. The NIH Office for Protection from Research Risks has interpreted “live vertebrate animal” to apply to avians (e.g., chick embryos) only after hatching.” Since our work does not utilize hatched chicks, no Institutional Animal Care and Use protocol for this work is necessary.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Partly\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nNot applicable\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes",
"responses": [
{
"c_id": "9078",
"date": "12 Dec 2022",
"name": "Lisa Taneyhill",
"role": "Author Response",
"response": "We thank Dr. Wray for their insightful comments on the manuscript. We have responded to each point below and have made necessary updates to the manuscript, where applicable. All Reviewers raised issues with the lack of quantification and statistics in the manuscript. To address this, we have now quantified GFP-positive puncta in our in vivo experiments. This was made possible by changing antigen colors in the images (as per Dr. Forni’s suggestion) and using a software program (Squassh plugin, FIJI) to quantify GFP-positive cells or regions between cells. Our data reveal a statistically significant increase in the number of GFP-positive regions in the Cad7 GFP1-10 + N-cad GFP11 electroporated embryos vs. the Cad7 GFP11 + N-cad GFP1-10 electroporated embryos (p = 0.014). We discuss possible reasons for this in the Discussion section, speculating that the reduced number of GFP-positive regions in the latter could be due to the general larger size of the N-cad GFP1-10 plasmid, potentially precluding robust expression of this construct, and/or intrinsic differences in the ability of neural crest and placode cells to transcribe and translate expression constructs. Although it was not possible to distinguish individual GFP-positive puncta/regions in the cell culture experiments, we have reported the percentage of GFP-positive cells in this assay, along with statistical comparisons across treatments. While there is no statistically significant difference in the number of GFP-positive cells after transfection of homophilic split GFP constructs (i.e., Cad7 GFP1-10 + Cad7 GFP11 or N-cad GFP1-10 + N-cad GFP11), we note a statistically significant increase in the number of GFP-positive cells in Cad7 GFP1-10 + N-cad GFP11-transfected cells vs. Cad7 GFP11 + N-cad GFP1-10-transfected cells (p = 0.013). These findings are in keeping with our in vivo results and have all been incorporated into the revised manuscript as part of new Table 1. We appreciate the suggestion from Dr. Wray to compare the results from our sequential electroporation experiments of cadherin split GFP construct to those obtained after co-electroporation of N-cad GFP1-10 + N-cad GFP11 into placode cells. We have performed this experiment and quantified the number of GFP-positive cells, noting that such homophilic, cis interactions are more robust than those observed in our sequential electroporation experiments. These data are now provided as new Figure 9 and Table 2. As per the suggestion of both Dr. Forni and Dr. Wray, we have placed the antigen names for new Figures 7-9 on a solid background. In addition, we have included the DAPI staining for the in vivo GRASP experiments to better visualize cell bodies and GFP detection. We thank Dr. Wray for providing suggestions for text modifications for the Introduction and Ethical Approval sections and have made these changes in the revised manuscript."
}
]
}
] | 1
|
https://f1000research.com/articles/11-741
|
https://f1000research.com/articles/11-1487/v1
|
12 Dec 22
|
{
"type": "Research Article",
"title": "Sense of personal agency towards mitigating the threat of antibiotic resistance: a focus group study with parents of children under 5 years old, conducted mid-pandemic",
"authors": [
"Becky McCall",
"Andrew Hayward",
"Michael Wilson",
"Gill Forbes",
"Laura Shallcross",
"Andrew Hayward",
"Michael Wilson",
"Gill Forbes",
"Laura Shallcross"
],
"abstract": "Background: Most antibiotic prescribing occurs in primary care, largely in children under 5 years old, and often inappropriately. This study investigated knowledge, attitudes and behaviours (KABs) towards common childhood infections, antibiotic use and antimicrobial resistance (AMR), among parents of children under 5 years old. The concept of individual sacrifice (forgoing antibiotics—a selective pressure for AMR) to mitigate future societal risk of AMR and how the COVID-19 pandemic shaped views were explored. Methods: This qualitative study included three, one-hour, virtual focus groups with mothers from parenting networks across inner-city London and semi-rural England, held mid-pandemic (2020). All had ≥1 child <5 years old. The Framework Method of analysis was used. Parents' KABs towards antibiotic use/AMR formed the primary outcome, with emphases on their sense of personal agency towards mitigating the threat of AMR for society, plus how the pandemic influenced views on infection prevention and care. Results: Fourteen mothers (groups of six, four, four) participated, with mixed ethnicities, education and employment status. Parent perceptions of their individual child's immediate need for antibiotics outweighed concerns for any possible future threat of AMR to society. Four key themes were identified: uncertainty around symptoms; impact of socio-cultural background on KAB; poor understanding of how antibiotics/AMR work; and opportunities within the doctor–patient dialogue to shape mindset around AMR. The pandemic influenced views across themes. Conclusion: Parents prioritising their child's perceived, immediate, individual 'need' for antibiotics over any future impact of AMR on society highlights a continuing need to engage parents in how to mitigate AMR through appropriate antibiotic use, reducing threat to both their child and others. Framing point-of-care dialogue around antibiotic use/AMR in the present (versus future), drawing on pandemic insights and tailoring according to nuanced socio-cultural influences, may encourage a greater sense of personal agency towards taking action to mitigate antibiotic resistance.",
"keywords": [
"antibiotic*",
"'antibiotic resistance'",
"parent*",
"'focus group'",
"GP",
"COVID",
"prevention",
"infection."
],
"content": "Introduction\n\nAmong the multiple stakeholders tackling the global challenge presented by antimicrobial resistance (AMR), the general public, including the parents of children aged under 5 years old, the specific population in this study, play a central role.\n\nThis study refers specifically to antibiotic resistance in the UK, where around 80% of antibiotic prescribing occurs in primary care. Data show that up to 23.1% of such prescriptions are considered inappropriate by experts.1\n\nApproximately 97% of preschool children consult a doctor at least once, most often for uncomplicated respiratory tract infections (RTIs) that mostly do not benefit from antibiotics.2 This suggests significant scope for improvement in antibiotic prescribing in this age group.3,4\n\nUK data from 2013 showed that 'patient knowledge, beliefs and attitudes may drive excessive antimicrobial use', including through patient influence during consultations.5 Prior to the COVID-19 pandemic, gains were starting to be made, with a 13.6% reduction in antibiotic prescribing in England between 2014 and 2018.6 However, despite this progress, inappropriate use/prescribing of antibiotics persists, with 2022 data showing wide variation across practices, highlighting that continued efforts to effectively counter this are needed.7,8\n\nThe past decade has seen an emphasis on improving antibiotic stewardship alongside public health campaigns to improve public understanding of AMR.9–11 How this has translated into parental perceptions around antibiotics/AMR formed one strand of this study—both generally, and in the light of the COVID-19 pandemic, during which this study was conducted.\n\nNovel communication methods including the use of personalised, educational patient information leaflets together with dialogue-oriented, rather than prescription-oriented, approaches have yielded a reduction in both antibiotic prescribing and reconsultation rates.12–14\n\nHowever, to help optimise the patient-centric nature of this approach, a more nuanced harnessing of the socio-cultural drivers of knowledge, attitudes and behaviours (KABs) towards antibiotic use and AMR, within the doctor–patient dialogue around AMR, may impact personal attitudes, social norms and perceived barriers to responsible antibiotic use – an articulated objective of the UK Government.5,15\n\nWith respect to socio-cultural influences on KABs of AMR, this study was conducted in the middle of the most significant infectious disease pandemic for a century—COVID-19. As such, consideration was given to the influence of the pandemic on views around infectious disease prevention and management.\n\nThe fundamental concept of adopting an individual sense of personal agency, for example, foregoing an antibiotic (for non-serious infections) to help mitigate AMR for future societal benefit provided this study with a novel lens though which to understand parent perceptions. This is an often-used concept that frames AMR as a humanitarian crisis potentially leading to 10 million deaths by 2050, but this may precipitate a sense that personal action to avert this destiny is beyond an individual's reach, as echoed in another study, with parents, 'unsure as to how they could reduce antibiotic resistance themselves as the problem was part of a “much bigger” picture’.16,17\n\nThis study aimed to obtain a snapshot of the perceptions and behaviours (including parent perceptions of doctor–patient interactions) around antibiotic use and AMR of parents with respect to their children, and to interpret these through the novel lenses of both a sense of personal agency towards mitigating the threat of AMR for individual as well as societal gain, both now and in the future, and, uniquely, within the setting of the COVID-19 pandemic.\n\n\nMethods\n\nThis qualitative study included three focus groups comprised of parents of children aged under 5 years old. Originally planned as an in-person activity, pandemic restrictions required an amendment to the original NHS Health Research Authority (HRA) ethical approval (REC number 19/LO/1820, HRA approval February 2020). The duration of each focus group protocol was adapted to approximately one hour, deemed optimal for a virtual focus group.18,19\n\nParticipants were recruited using a purposive sampling method. Two easily reachable geographical areas (originally selected for in-person groups) were chosen providing a mix of inner-city urban (Islington, London; one group), and semi-rural (Hertfordshire; two groups), as well as diversity in ethnicity, educational attainment and employment status. Leaders of preschool parenting networks (employees of Islington Borough Council and Hertfordshire Community NHS Trust) facilitated recruitment through official social networks, for example Facebook groups or weekly meetings, as well as via parent champions who recruited both directly and through snowball sampling until sufficient numbers were reached.\n\nParticipants were provided with participant information sheets and electronic consent forms, along with an email introducing the study. Author BM answered any questions to ensure fully informed consent was given, and participants signed and filed the consent forms electronically, and received a £20 voucher for their contribution.\n\nParents’ basic demographic data were gathered, including employment status, ethnicity and parity. Participants were asked to respond in their capacity as parents.\n\nThe topic guide listing a priori themes was jointly developed by the researchers, including two with experience in clinical and public health issues related to antibiotic use/AMR (authors LS and AH). It was structured to provide a snapshot of current understanding around antibiotic use, and both the threat felt (if any), and the sense of responsibility participants held towards their children, and others, in mitigating the emergence and spread of AMR. Topics addressed infection self-care, antibiotic-seeking KABs, the nature and consequences of AMR including sense of individual agency in mitigating AMR for societal good, and COVID-19 influence on views towards infection prevention and management. After piloting by two parents of young children, slight modifications were made to enhance the lay-friendly appeal of content.\n\nAuthor BM was the focus group facilitator, and only member of the research team attending the sessions. Secure and encrypted MS Teams video conferencing technology was used to collect and record the focus group data. All participants contributions were transcribed and anonymised.\n\n(See Table 1 for a topic guide summary; see extended data for full topic guide).\n\n\n\n1. Approach to treating illness (infections) in your child (including the influence of COVID-19 pandemic on infection prevention and management)\n\n‐ For which illness/es and symptoms would you seek healthcare professional (HCP)/doctor advice?\n\n‐ Who and what services have you contacted, e.g. out of hours, walk-in centre?\n\n‐ How would you provide home care, and over the counter advice/treatment (prior to approaching a doctor)?\n\n‐ Have your children received recommended childhood vaccinations?\n\n‐ Do you consider vaccination an important preventive measure against infectious disease?\n\n‐ How has COVID-19 influenced your views towards infection prevention and management including the potential for COVID-19 vaccination (both for you and your children)?\n\n2. Antibiotic use: experience of your child (or you secondarily)\n\n‐ When did you last see an HCP/clinician in a situation when you thought your child might need an antibiotic (and either received an antibiotic or not)?\n\n‐ Have you ever been refused antibiotics and how did that conversation play out between you and the clinician?\n\n‐ Does your doctor ever initiate a discussion around the need for antibiotics, and, if so, does s/he refer to the downsides of antibiotic use, e.g. resistance or other?\n\n‐ Have you ever sought antibiotics from somewhere other than your GP, and if so, why (e.g. walk-in centre, out-of-hours centre, NHS111, online)?\n\n‐ Do you consider that antibiotics can be harmful as well as beneficial? Expand on harms versus benefits.\n\n3. Antibiotic resistance and perceived threat, if any, from AMR\n\n‐ What do you understand by the term ‘antibiotic resistance’ (AMR)?\n\n‐ How does antibiotic resistance develop (including any mechanistic understanding)?\n\n‐ To what extent do you feel personal risk (or risk to your child) from a drug-resistant infection/AMR?\n\n4. Societal consequences of antibiotic resistance and responsibility/agency to mitigate effects\n\n‐ Do you believe your personal actions towards antibiotic use can influence development of AMR and have an impact on your/your child's health and that of the wider public in the long-term? (Possible parallel with potential COVID-19 vaccination to prevent community spread.)\n\n‐ What are the direct and indirect consequences of AMR for your child as an individual but also for society in the longer-term?\n\n‐ People talk about ‘an antibiotic crisis’, what does this mean to you?\n\n‐ Do you think individual (personal sense of agency) or governments/big organisations should take greatest responsibility for tackling the threat of AMR?\n\nTranscripts of the recorded discussions were entered into Computer-assisted qualitative data analysis software (CAQDAS, NVivo Version 12 (RRID:SCR_014802)) to organise the data. Alternative open access software to NVivo exist for example, Taguette or RDQA.\n\nQualitative analysis followed the established Framework Method. This entailed a stepwise process of data familiarisation; mostly inductive analysis consisting of line-by-line coding/sub-coding (using NVivo) and grouping into categories broadly aligned (but adapted to reflect conceptual relationships between comments) with the a priori topics in the topic guide, and this effectively created an analytical framework. This was applied across all transcripts to compare cases (participant comments) both within focus groups and across focus groups until no new codes were generated (data saturation point). By charting the data into a ‘framework matrix’ comprising codes in columns, and cases in rows (see extended data for a table of codes and descriptions, and for an example of the framework matrix), thematic analysis was conducted. This involved the generation of sub-themes initially, and then key themes (synthesis across sub-themes). Ultimately, insights (possible unarticulated explanations) were derived from reviewing the matrix and drawing connections within and between participants and categories to facilitate higher order interpretation, according to a process of thematic analysis.23–26\n\nCoding and themes were checked for consistency and reliability with two co-authors (LS and AH) and to balance any reflexivity of BM, a mother of young children, in analysis of the data. Such similarity may be considered a bias, but also a benefit that may enhance rapport and the richness of data obtained.27\n\nConsolidated criteria for reporting qualitative research (COREQ) were followed as closely as possible in the reporting of this research.28 In accordance with these guidelines, lead author BM attended training in both facilitating a focus group and conducting qualitative data analysis. BM had no relationship with participants prior to the study and the participants understood the research formed part of BM's doctoral research.\n\nFour members of the public were consulted in designing the topic guide and provided input on content and style of questions, as well as proofing.\n\n\nResults\n\nA total of 14 parents participated in three virtual focus groups between August and October 2020, with a topic guide adapted from the in-person to the virtual format. The adapted virtual format shortened the focus group duration and the last section on public health campaigns was insufficiently answered to warrant inclusion. Two participants dropped out: one due to technical connection issues, and one due to a hospital appointment. One other was a medical doctor and it was decided this potential participant would be too conflicted to include in this study that sought views from parents who were members of the lay public.\n\nMost participants (10 out of 14) were aged 30–40 years and White British (nine out of 14); all were female and with at least A level or equivalent of educational attainment, and all had at least one child under five years. Most (11 out of 14) children had received antibiotics at least once: eight out of 14 from a GP, and six out of 14 from secondary care or an NHS walk-in centre (see Table 2).\n\nIn accordance with SAGER guidelines for reporting sex and gender information in studies, this study was designed to recruit parents, without gender discrimination. Only mothers volunteered to join the focus groups.\n\nThe process of analysis using the Framework Method generated 13 codes, divided into four categories that emerged from the data (see extended data for a table of descriptions according to categories and codes; and a table of categories aligned by their codes and sub-codes).\n\nAnalysing each transcript according to these codes and selecting verbatim illustrative of them generated insights including drivers of KABs around AMR. Synthesis across the three focus groups generated four key themes, which are presented alongside their sub-themes, codes, and categories/a priori topics in Table 3.\n\nKey themes 3 and 4 are most novel, while some aspects of key themes 1 and 2 are relatively new but largely reinforce findings from other studies. Table 3 shows the relationship of the original a priori topics (from the topic guide) to sub-themes and key themes (sub-themes and key themes were derived from inductive analysis).\n\nKey theme 1: Uncertainty around the management of symptoms and severity of childhood infection, including when to consult a GP (possibly for antibiotics), including the influence of the pandemic on views around prevention and management of infection.\n\nMost parents remarked on their uncertainty about when the severity of their child’s symptoms warranted a doctor consultation, possibly for antibiotics. Many said they home-managed symptoms for around three days before seeking medical help. Mothers with older children (compared with first-time mothers) suggested greater confidence in managing their child's illness at home for longer.\n\nOne mother recalled giving her 10-month old 'Calpol' if he had a fever, but if she felt his ‘heart rate was up and he wasn’t feeding properly’ then she would contact a doctor. (Focus Group (FG)2, participant (p)3)\n\n'I didn’t take her for a few weeks because I was like, “Oh she’s teething, she’s got a bit of a cold, she’s just gone back to nursery after lockdown”.’ (FG2, p4)\n\n‘As a first-time mum, you’re a little bit more nervous and apprehensive about things, having not done it before.’ (FG2, p3)\n\nSuch comments highlight the need for improved public/patient engagement around which symptoms and severity justify contacting a GP (potentially seeking antibiotics).\n\nSometimes non-medical pressures are implicated—economic (employment-related) factors were alluded to.\n\n'It’s a bit selfish but I find it quite hard to get her to the doctor, because you have to take time off work, I’ve got to get her out of nursery.' (FG2, p4)\n\nThe COVID-19 pandemic profoundly influenced people’s attitudes and behaviours towards infection prevention and control, with constant public health messages around handwashing and other SARS-CoV-2 mitigation measures. Community antibiotic prescriptions fell during this period.29\n\n‘Covid has raised my awareness of infection, and actually, not everything can be treated by antibiotics, and sometimes, you’ve just got to ride things out depending on the severity of your child’s illness.’ (FG1, p6)\n\nVaccination, generally, was also raised as a measure to prevent infection and was particularly topical at the time, with potential COVID-19 vaccinations being debated publicly.\n\nTable 4 lists examples of verbatim relevant to this key theme.\n\nUncertainty in the management of symptoms and severity of childhood infection, including when to consult a GP (possibly for antibiotics), including the influence of the pandemic on views around prevention and management of infection.\n\n\n\n• Uncertainty about when symptoms (and severity) justify GP care, and home management of symptoms.\n\n‘I just check out the NHS website as a first point of call, if I don’t believe that the symptoms appear that serious.’ (FG1, p5)\n\n‘… it’s important to stay healthy, by having a good diet … I take probiotics - so vitamins, probiotics to avoid infections as well.’ (FG1, p4)\n\n‘With my little one I barely gave him any antibiotics. I’ve changed my approach I would say. I’ve been in the UK for 15 years now.’ (FG3, p1)\n\n‘… I have four children, oldest 15, youngest three, so, with something like tonsillitis, they would have to have antibiotics, but I would go to the GP’. (FG2, p2)\n\nAnother parent misinterpreted messages around AMR and avoided the GP, resulting in her child being admitted to hospital. She recalled that had she, 'had just taken the antibiotics in the first place then I [her child] wouldn’t have been'. (FG 2, p1)\n\n\n\n• Parents were more cautious with their child than themselves (sense of child's vulnerability).\n\n‘ … when I was prescribed the [antibiotic] cream for my child I did not hesitate, I was gonna do it.' and ‘… for my daughter I’d always finish the course.’ (FG3, p4)\n\n\n\n• Parents who took their child to an out-of-hours/walk-in/emergency centre most often received an antibiotic prescription.\n\n‘Almost any time I ended up at out-of-hours or A&E, I got antibiotics, even if I was told they weren’t needed only a few hours earlier.’ (FG1, p3)\n\n‘… we delayed and kept him at home for a while and then went to an out-of-hours [service], and they just instantly prescribed antibiotics. I think when it’s a chest infection, I’d rather have antibiotics to clear it up, I think because when it’s something to do with breathing …’ (FG2, p1)\n\n\n\n• Vaccination to prevent infection was mostly supported.\n\n‘My mum was an anti-vaxxer so I wasn’t vaccinated until I was 18 when I could make the decision myself for that. As a result of not having those vaccines, I’ve got cancer-causing HPVs and I need so many checks—I’m angry because of that, so, with [daughter’s name], she was going to have every vaccine possible, so she doesn’t have the same experiences that I’ve had.’ (FG1, p4)\n\n‘I’m having more fruit and veg, the same for my kids, I’m really not into having a vaccine [with respect to COVID-19], or having some antibiotics, I know that it wouldn’t resolve the problem, because we don’t know what it is, and I don’t think they know how to cure it anyway.’ (FG3, p1)\n\n\n\n• COVID-19 has heightened concerns about catching infections (SARS-CoV-2 or other).\n\n‘It’s hospital but if I can do anything else before I take them to a place where other sick people go [response to COVID pandemic discussion], I’ll do it.’ (FG1, p3)\n\n‘Had he been born outside of Coronavirus times, I probably wouldn’t have been as concerned about him catching infections … I wouldn’t have been as worried about it as I am now, and I think that’s the same with antibiotics.’ (FG 3, p1)\n\nKey theme 2: Background including cultural, social, generational, familial and habitual factors influence KABs towards antibiotic use/AMR\n\nSubtle and often unarticulated factors, including generational, familial, cultural, habitual, geographical and social, often influence views towards healthcare, including antibiotic use/AMR, as well as parent/patient expectations from the doctor–patient consultation.\n\nOne mother who grew up in Somalia said they expected to receive medication upon visiting the GP, effectively as a validation of their illness, ‘be it antibiotic or medicine or anything'.\n\nAnother mother said that antibiotics were habitually prescribed in her parent’s home country, France.\n\n‘… in France when you get sick, it’s antibiotics … . sometimes if my kids were really, really sick I would buy medication from France and bring my medication here. My mum, she sends me parcels [with antibiotics].’ (FG3, p1)\n\nOlder generations lived in a time when antibiotics were considered a cure-all.\n\n‘I think it’s difficult for the older generation that think that antibiotics are the answer to everything.’ (FG3, p4)\n\nA mother from Spain said antibiotics were much more available there than in the UK, and, in her opinion, there was a lack of universal consensus around antibiotic stewardship.\n\n‘In the UK, antibiotics for urinary tract infections [UTIs] require antimicrobial pharmacist approval, … whereas, in Spain, they just sort of hand [them] out left, right and centre.’ (FG1, p4)\n\nUnderstanding more about the socio-cultural drivers and experiences underpinning an individual's KABs towards antibiotic use may illuminate novel, more nuanced, avenues for formulating and communicating AMR messages.\n\nCertain areas of the UK with greater socio-cultural diversity may benefit most from incorporating a more nuanced and tailored approach to the doctor–patient dialogue around antibiotics/AMR.\n\nKey theme 3: Greater understanding of how antibiotics work, and how AMR develops, may impact perception of threat to the individual and society, and consequently individual sense of agency/responsibility towards mitigating AMR\n\nOverall, lay understanding of how antibiotics and AMR work was mixed but largely limited. Most parents knew that antibiotics fight bacteria, not viruses, with one mother understanding that antibiotics can be either narrow or broad spectrum. That antibiotics can target good bacteria too was mentioned. ‘… you don’t know what’s getting killed basically …’ (FG2, p4)\n\n'It's a bit like the bug becomes stronger, a bit like we’re seeing with Covid and they’re talking about all these different strains, and it mutates – AMR, that’s what I understand.' (FG2, p1)\n\nFour participants believed that antibiotic resistance meant the body became tolerant to antibiotics.\n\n'Your body changes over time, something that worked for you once, might not work for you again.' (FG1, p5)\n\nThe relatively common misunderstanding that resistance implies the body becomes tolerant to antibiotics, rather than resistance being a feature of the infecting bacteria, may be instrumental in the sense of threat individuals perceive from AMR, and may help to explain why many people see AMR as a threat not associated with them personally, but only with others who use antibiotics frequently. Some parents felt the threat of AMR was not very present in their lives today.\n\n‘I think it is on the risk scale but it’s not something that I worry about every day.’ (FG1, p1)\n\n‘I had always thought, like you said, it’s something that will happen in the future or something that’s coming but it’s not really [here now].’ (FG2, p4)\n\nAMR is often framed as one of society's most serious global humanitarian crises, and, as such, participants were asked whether they would personally forego an antibiotic now to prevent future widescale AMR adversity for society. Despite some parents recognising this, all parents said that the immediate threat of infection to their individual child’s health took priority and that they would seek antibiotics if it promised to help their child.\n\n‘Do I really want him to become antibiotic resistant? But equally I don’t want him to get infections.’ (FG2, p1)\n\nParents reflected on securing a future free of AMR and the benefit it offered to their child in preference to society. Together, these comments suggest that focusing on benefit to the individual rather than society would potentially resonate more with parents.\n\nTwo parents highlighted that the lack of an effective treatment for COVID-19 (summer 2020) echoed the nature of AMR, whereby antibiotics are ineffective against infection.\n\n‘There’s a bit more awareness that not all illnesses have a definitive treatment, and that antibiotics are [not] the cure-all as some people believe.’ (FG1, p1)\n\nThe mitigation measures taken by individuals to reduce societal spread of COVID-19, e.g. vaccination, isolation, masks and social-distancing, reflect the narrative pertaining to AMR where individual action now (foregoing an antibiotic) helps to avoid future adverse effects for society (emergence of AMR later).\n\nMost parents said they would have a COVID-19 vaccination, when they became available. Of note, the groups were held mid-pandemic and the threat of COVID-19 felt real, such that the risk of future adversity (COVID-19) was considered serious enough to warrant the small inconvenience and/or discomfort of vaccination. By extension, this might highlight the value of imparting knowledge about the real and immediate threat of AMR and how to avert it when people are primed to receive such information, for example, at the point of care, when parents consult a GP with a sick child.\n\nTable 5 lists examples of verbatim relevant to this key theme.\n\nGreater understanding of how antibiotics work, and how AMR develops, may impact perception of threat to the individual and society, and consequently individual sense of agency/responsibility towards mitigating AMR.\n\n\n\n• There was some understanding around how antibiotics work but also a need for further clarification for most participants.\n\n‘… they inject something, like into the body, in a small amount so then the body can fight against it, something like this.’ (FG3, p1)\n\n‘… some antibiotics can be … broad spectrum. They will kill more bugs than other ones …’ (FG1, p1)\n\n\n\n• Inappropriate use of antibiotics relates to animal use as well as human.\n\n‘...like the chicken, they always inject with antibiotics, so actually we’re taking them without realising we’re having antibiotics …’ (FG2, p1)\n\n\n\n• A mixture of confusion and some understanding around how AMR develops\n\n‘… because we’ve been taking the same type of medication it’s not going to have an effect on your body, because your body knows about this treatment … when you have taken them frequently, your body can build up a tolerance to them so they don’t necessarily fight off the bacteria as effectively as they might have done the first time you were prescribed them.’ (FG2, p1)\n\n‘… if your immune system isn’t given the opportunity to work and to attack both the build-up of antigens and antibodies in your own body, that will mean you are less good at fighting other diseases in the future.’ (FG1, p1)\n\n\n\n• There was some confusion around whether the threat posed by AMR was a real threat today, or a possible threat for the future.\n\nOne parent said she was ‘terrified’, and that AMR is ‘extremely widespread’. Using COVID-19 as a reference, she added, ‘COVID-19 is bad, but I really am a lot more concerned by AMR’. (FG1, p3)\n\n\n\n• COVID-19 highlighted to some parents that not all infections have an effective treatment.\n\n‘I think people are a bit more aware of the untreatable diseases and ones that are viruses as opposed to other infections.’ (FG1, p1)\n\n\n\n• Threat of a current infection was more real than threat associated with any possible resistant infection in the future.\n\nOne child was ill with a urinary tract infection. ‘… it’s the lesser of two evils … I guess you’ve just got to deal with what you’ve got to deal with at the time haven’t you?’ (FG2, p4)\n\n‘I’d be more worried about my son than I would be about myself. The way I perceive it is that it’s going to happen over time, or maybe to me when I’m older.’ (FG3, p3)\n\n‘I’ve lived so much in a bubble where antibiotics have cured everything, for me personally, and people I know, I’ve not really thought about it.’ (FG2, p1)\n\n‘I do look at it as more of a personal problem with individual bacterial resistance rather than being a kind of, do it for the community, so I take it as a personal kind of thing for me and my son.’ (FG2, p3)\n\n\n\n• Many did not consider AMR a real and current danger. Some parents believe science will find an answer.\n\n‘It’s not really crossed my mind that there wouldn’t be a solution to a bacterial infection.’ (FG2, p1)\n\nKey theme 4. Strength of the doctor–patient dialogue serves as an opportunity to effect change in KABs relating to antibiotics/AMR at the point-of-care.\n\nParticipant comments in this study indicate that the primary care consultation setting is conducive to a constructive interaction around the risks and benefits of antibiotics and AMR.12–14 As such, these data both add value to, and reflect, prior reports of antibiotic stewardship leading to a reduction in antibiotic prescribing in primary care over recent years (excepting anomalies related to pandemic prescribing).6,30 Such constructive dialogues nurture improved GPpatient trust and enhanced compliance, potentially reaping short and longer-term benefit.\n\n‘Now they are clearer when they say it, so I trust my GP.’ (FG3, p1)\n\nThat clinicians have a valuable role to play in alleviating misunderstanding around antibiotics, and AMR was widely accepted by parents, most of whom said that they followed the doctor’s advice on antibiotic use.\n\n‘If they think it [an antibiotic] 's needed they will prescribe them, so I tend to go on what they say with babies.’ (FG2, p1)\n\nGP-led antibiotic stewardship efforts and education of specific knowledge at the point-of-care was important to participants.\n\n‘My first thought would be if a doctor says, “We can get through this without antibiotics”, then that would be my preference. I rely on their opinion.’ (FG2, p3)\n\nHowever, one parent noted that the differing opinions of doctors within practices can be confusing, emphasising the importance of consistency across healthcare points of contact with patients.\n\n‘It was just that conflicting advice of, if one doctor … [prescribes it] but then a second doctor [says] … they wouldn’t have prescribed it …’ (FG2, p3)\n\nAnother mother's comment illustrated how a parent’s instinct about their child sits alongside, and possibly carries equal weight to, a GP’s advice, reinforcing the need to move beyond a more conventional telling or provision of advice by doctor to patient, to the holding of a constructive two-way exchange. A more nuanced dialogue around antibiotics use/AMR may better address any barriers to adopting a mindset where antibiotic use reflects concerns around AMR.\n\n‘For my son, I know him better than any doctor does.’ (FG2, p3)\n\nTogether with key theme 2 (socio-cultural influences on KABs), the doctor–patient consultation setting may be optimised with a more personalised approach to facilitating and addressing any barriers to appropriate antibiotic use/AMR.\n\n\nDiscussion\n\nA central concept underpinning this study is taking action at an individual level to mitigate the threat posed by AMR to society – projected at around 10 million deaths from AMR by 2050 if no action is taken (2016 UK AMR Review). However, evidence from this study suggests that, despite parents acknowledging AMR as a possible future problem for society (including for their child), the threat is not considered real or relevant enough to justify the sacrifice of their child foregoing antibiotics, and the immediate need to seek medical help/antibiotics is the overriding concern. This, in itself, is not entirely surprising, but it does serve to highlight that framing messages around antibiotics/AMR in terms of risks and benefits to their individual child now, versus risks and benefits to society in the future, may resonate more with parents and possibly the wider public too.\n\nThis trade-off between individual versus societal benefit is reflective of the dynamics that sometimes underpin attitudes towards COVID-19 vaccination, the benefit of which is often more apparent at a population level, especially in younger people (≤40–50 years) who are at lower risk of severe disease. At the time of the study, such vaccinations were not publicly available, but they were widely discussed, including risks and benefits to individuals and society. In effect, the COVID pandemic may have attuned collective thinking around the concept of individual versus societal benefit with regards to widescale health. Few members of today's UK population have previously had to make the personal sacrifices that they did during the pandemic, e.g. isolating or restricting socialising to primarily benefit population health. As remarked upon by participants, the early months of COVID also highlighted the lack of effective treatment, a scenario paralleled by attempts to manage a (multi-) drug-resistant infection.\n\nOur study also reinforces that the fundamental misunderstandings relating to the biological basis of AMR persist among parents and may partially explain the finding that most parents do not perceive the relevance or benefit of harnessing a personal sense of responsibility towards mitigating AMR.8,31\n\nFinally, insights on the socio-cultural drivers of KABs around AMR are notable and suggest a more personalised doctor–patient dialogue around AMR may be more constructive. A 2018 WHO survey of antibiotic awareness campaigns showed that, to make further progress, campaigns should move towards locally adapted communication. Just as public health campaigns need to localise, so patient communication at the consultation level needs to be tailored to the individual.32\n\nOur data build on those already published on public perceptions of antibiotic use and AMR,8,31 and will inform future research on how to enhance the relevance and impact of such communication for both society and individuals in their relationship with antibiotics.\n\nAddressing the topic of parental KABs around antibiotic use and AMR through the concept of individual agency to avert AMR is novel but notably builds on a growing body of relevant, associated literature.\n\nSimilar to our findings, another recent study observed that, ‘parents found it difficult to interpret symptoms and signs’, and, ‘[…]need better information and support to manage their child’s illness at home’, adding further emphasis to the continuing need for intervention in this respect.33 Likewise, the perceived vulnerability of children, has been documented elsewhere.8\n\nPrevious studies with families attest to long-held misunderstandings about AMR, for example, parents perceiving that they are at low risk because they infrequently use antibiotics, and that the 'body becomes immune to them [antibiotics]'.2,17 Our study reinforces that such misunderstandings persist and suggests that more novel and potentially more effective means of engagement are needed.\n\nA 2016 study noted the complexity of the doctor–patient interaction and 'interplay of care seeking'.34 Our study reinforces and importantly builds on this by noting the nuances of parental background influences on KABs, suggesting that recognising, for example, ethnic, generational and country-of-origin differences may enhance the effectiveness of the doctor–patient dialogue.\n\nPrior studies have referred to the benefits of the point-of-care setting to optimise AMR engagement, but more research is needed on how this may manifest in practice.12–14,17,35,36 Pointedly, Cabral's 2016 study concluded the need for interventions that reduce antibiotic prescribing 'to address within-consultation communication, prescribing behaviour, and lay beliefs simultaneously'.35 Our study advances this with specific aspects of 'how' these within-consultation communication and lay beliefs may be approached and leveraged to further shape mindset towards antibiotic use/AMR, for example, focus on the risks and benefits to the individual of judicious antibiotic use. It also suggests that making this within-consultation communication more real and relevant to the individual's immediate situation may be more impactful.\n\nParticipant recruitment comprised a broad cross-section of parents from South-East England, which provided a rich demographic diversity due to this area having a high population density, of widely variable backgrounds, across both urban and semi-rural settings. However, there may have been some selection bias, with most parents having very good levels of education and employment, which is largely unrepresentative of the general UK population.\n\nPandemic restrictions means the virtual (at-home) setting may be viewed as both a strength and a limitation. The format facilitated attendance by parents whose childcare commitments might have prevented attendance at an in-person group. However, this also led to some unavoidable parenting distractions.\n\nParticipant numbers were small, and it is not possible to draw generalised conclusions based on the comments of only 14 participants, but insights would draw strength in combination with findings from other studies, with our findings reinforcing and building upon prior findings.\n\nThe virtual format also meant the group was shorter in duration, and that the interactions between group members were less natural than an in-person group. In addition, resource constraints and the virtual format meant non-verbal group dynamics could not be recorded.\n\n\nConclusions\n\nThese observations, in combination with a public engaged with the risks of infectious disease by the COVID-19 pandemic, suggest that clinicians and policymakers may frame messages around antibiotic use/AMR with an emphasis on the here and now (ideally delivered at the point-of-care), and drawing on relevance to their individual child at the present time, rather than referring to the impact of the future possibility of AMR for society. Tailor-making messages that are real and relevant to the individual would also benefit from a more nuanced approach that recognises the influence of an individual's multi-faceted socio-cultural background. Table 6 provides conclusions and recommendations based on this study.\n\nNovel insights on how the pandemic has shaped participant views of infections, antibiotics and AMR illustrate how tapping into an individual's sense of immediate need including risks and benefits of antibiotics may better resonate (and possibly motivate too) with concepts such as foregoing an antibiotic to benefit both the individual and society, in a similar way to most individuals undergoing COVID vaccination to benefit not only themselves, but also, possibly more so, those people around them. Effectively, AMR messaging needs to leverage individual benefit to maximise societal gain. It may also draw on the concept that healthcare does not always have an effective treatment, as seen with management of COVID and with AMR.\n\nFuture research may investigate how to draw on these findings of individualisation and contextualisation, as well as timing and setting to both improve understanding and tailor meaningful communication that resonates with the parents of young children and the public more widely, to optimise parental sense of agency towards mitigating the threat of AMR.\n\n\nEthical approval\n\nNHS Health Research Authority (HRA) ethical approval (REC number 19/LO/1820, HRA approval February 2020.\n\n\nConsent\n\nWritten (electronic) informed consent for publication of the participants' details was obtained from the participants.",
"appendix": "Data availability\n\nOpen Science Framework (OSF): Underlying data for ‘Sense of personal agency towards mitigating the threat of antibiotic resistance: a focus group study with parents of children under-5 years, conducted mid-pandemic’. https://doi.org/10.17605/OSF.IO/VN3X5. 37\n\nThe project contains the following underlying data:\n\nED 1: Topic guide (full version).\n\nED 2: Table of descriptions according to categories and codes.\n\nED 3: Categories and their codes (and sub-codes).\n\nED 4: Example of the framework matrix for category 4: Consequences of antibiotic resistance.\n\nED 5: Study Protocol.\n\nED 6: COREQ guidelines checklist\n\nData are available under the terms of the Creative Commons Zero “No rights reserved” data waiver (CC0 1.0 Public domain dedication).\n\nFirst author: Becky McCall becky.mccall.18@ucl.ac.uk\n\n\nAcknowledgements\n\nAll participants. Islington Council); and Mark Whiting (Hertfordshire Community NHS Trust) and colleagues (Hertfordshire Community NHS Trust) for help with recruitment.\n\n\nReferences\n\nSmieszek T, Pouwels KB, Dolk FCK, et al.: Potential for reducing inappropriate antibiotic prescribing in English primary care. J. Antimicrob. Chemother. 2018; 73: ii36–ii43. PubMed Abstract | Publisher Full Text | Free Full Text\n\nHalls A, Van’T Hoff C, Little P, et al.: Qualitative interview study of parents’ perspectives, concerns and experiences of the management of lower respiratory tract infections in children in primary care. BMJ Open. 2017; 7(9): e015701–e015708. Publisher Full Text\n\nStuart B, Brotherwood H, Van’T Hoff C, et al.: Exploring the appropriateness of antibiotic prescribing for common respiratory tract infections in UK primary care. J. Antimicrob. Chemother. 2020; 75(1): 236–242. PubMed Abstract | Publisher Full Text\n\nLittle P, Francis NA, Stuart B, et al.: Antibiotics for lower respiratory tract infection in children presenting in primary care in England (ARTIC PC): a double-blind, randomised, placebo-controlled trial. Lancet. 2021; 398(10309): 1417–1426. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMcNulty CA, Nichols T, French DP, et al.: Expectations for consultations and antibiotics for respiratory tract infection in primary care: the RTI clinical iceberg. Br. J. Gen. Pract. 2013 Jul [cited 2019 May 9]; 63(612): e429–e436. PubMed Abstract | Publisher Full Text | Free Full Text\n\nThomson K, Berry R, Robinson T, et al.: An examination of trends in antibiotic prescribing in primary care and the association with area-level deprivation in England. BMC Public Health 2020; 20(1): 1–9. Publisher Full Text\n\nVan Staa T, Li Y, Gold N, et al.: Comparing antibiotic prescribing between clinicians in UK primary care: an analysis in a cohort study of eight different measures of antibiotic prescribing. BMJ Qual. Saf. 2022; 31: bmjqs-2020-012108–bmjqs-2020-012838. PubMed Abstract | Publisher Full Text | Free Full Text\n\nPinder R, Sallis A, Berry D, et al.: Behaviour change and antibiotic prescribing in healthcare settings Literature review and behavioural analysis Public Heal. Engl. 2015.Reference Source\n\nBhattacharya A, Hopkins S, Sallis A, et al.: A process evaluation of the UK-wide Antibiotic Guardian campaign: developing engagement on antimicrobial resistance. J. Public Health (Oxf.) 2017 Jun 1; 39(2): e40–e47. PubMed Abstract | Publisher Full Text\n\nDepartment of Health, Department for Environment Food and Rural Affairs: UK Five Year Antimicrobial Resistance Strategy 2013 to 2018. Dep. Heal. Dep. Environ. Food Rural Aff. 2013; 43. Date accessed Nov. 2022.Reference Source\n\nDepartment of Health and Social Care: UK Government. The UK’s five-year national action plan. Policy Paper. 2019. Date accessed Nov. 2022.Reference Source\n\nDe Bont EGPM, Alink M, Falkenberg FCJ, et al.: Patient information leaflets to reduce antibiotic use and reconsultation rates in general practice: A systematic review. BMJ Open. 2015; 5(6): e007612. Publisher Full Text\n\nFrancis NA, Butler CC, Hood K, et al.: Effect of using an interactive booklet about childhood respiratory tract infections in primary care consultations on reconsulting and antibiotic prescribing: A cluster randomised controlled trial. BMJ. 2009; 339(7717): b2885–b2886. Publisher Full Text\n\nRoyal College of General Practitioners (RCGPs): Target Antibiotic Toolkit Hub. Version 1.0.Date accessed Nov. 2021.Reference Source\n\nMcnulty C, Owens R: Antimicrobial stewardship in primary care: developing a local action plan. (PDF of a PowerPoint presentation). 2015. Public Health England Primary Care Unit.Date accessed Nov. 2022.Reference Source\n\nNeill JO: Tackling Drug-Resistant Infections Globally: Final Report And Recommendations.May 2016. Date accessed Nov. 2022.Reference Source\n\nVan Hecke O, Butler CC, et al.: Parents' perceptions of antibiotic use and antibiotic resistance (PAUSE): a qualitative interview study. J. Antimicrob. Chemother. 2019 Jun 1; 74(6): 1741–1747. PubMed Abstract | Publisher Full Text | Free Full Text\n\nDos Santos Marques IC, Theiss LM, Johnson CY, et al.: Implementation of virtual focus groups for qualitative data collection in a global pandemic. Am. J. Surg. 2021; 221(5): 918–922.\n\nKite J, Phongsavan P: Insights for conducting real-time focus groups online using a web conferencing service. F1000Res. 2017; 6: 1–14. Publisher Full Text\n\nVanden Eng J, Marcus R, Hadler JL, et al.: Consumer attitudes and use of antibiotics. Emerg. Infect. Dis. 2003; 9(9): 1128–1135.\n\nMorgan DJ, Okeke IN, Laxminarayan R, et al.: Non-prescription antimicrobial use worldwide: a systematic review. Lancet Infect. Dis. 2011 Sep; 11(9): 692–701.\n\nKodish S: The Power of Narratives: A New Understanding of Antibiotic Resistance. Int. J. Commun. Jan. 2018; v. 12: p. 21. [S.l.]. 1932-8036.\n\nGale NK, Heath G, et al.: Using the framework method for the analysis of qualitative data in multi-disciplinary health research. BMC Med. Res. Methodol. 2013; 13(1): 1.\n\nBraun V, Clarke V: Using thematic analysis in psychology. Qual. Res. Psychol. 2006; 3(2): 77–101. Publisher Full Text\n\nRitchie J, Spencer L: Qualitative data analysis for applied policy research. Anal. Qual. data. 2010; 173–194.\n\nRitchie J, Lewis J: Qualitative Research Practice A Guide for Social Science Students and Researchers. SAGE Publications Ltd.;2003.\n\nRand JR: Inuit women's stories of strength: informing Inuit community-based HIV and STI prevention and sexual health promotion programming. Int. J. Circumpolar Health. 2016 Dec; 9(75): 32135.\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. Oxford:Academic;2007; 19: 349–357.\n\nGillies MB, Burgner DP, Ivancic L, et al.: Changes in antibiotic prescribing following COVID-19 restrictions: Lessons for post-pandemic antibiotic stewardship. Br. J. Clin. Pharmacol. 2022; 88(3): 1143–1151. PubMed Abstract | Publisher Full Text | Free Full Text\n\nNuffield Trust: Nuffield Trust Antibiotic prescribing. Prescribing patterns for antibiotics, both in England and internationally.September 2021. Date accessed: Oct. 2022.Reference Source\n\nWellcome Trust: Exploring the consumer perspective on antimicrobial resistance.June 2015. Date accessed Nov. 2022.Reference Source\n\nHuttner B, Saam M, Moja L, et al.: How to improve antibiotic awareness campaigns: Findings of a WHO global survey. BMJ Glob Heal. 2019; 4(3): 1–9. Publisher Full Text\n\nWoods CJ, Morrice Z, Francis NA, et al.: Parent and Clinician Views of Managing Children with Symptoms of a Lower Respiratory Tract Infection and Their Influence upon Decisions to Take Part in a Placebo-Controlled Randomised Control Trial. Antibiotics (Basel). 2021 Mar 28; 10(4): 356. PubMed Abstract | Publisher Full Text | Free Full Text\n\nHorwood J, Cabral C, Hay AD, et al.: Primary care clinician antibiotic prescribing decisions in consultations for children with RTIs: A qualitative interview study. Br. J. Gen. Pract. 2016; 66(644): e207–e213. PubMed Abstract | Publisher Full Text | Free Full Text\n\nCabral C, Ingram J, Lucas PJ, et al.: Influence of clinical communication on parents’ antibiotic expectations for children with respiratory tract infections. Ann. Fam. Med. 2016 Mar 1; 14(2): 141–147. PubMed Abstract | Publisher Full Text | Free Full Text\n\nButler CC, Gillespie D, White P, et al.: C-Reactive Protein Testing to Guide Antibiotic Prescribing for COPD Exacerbations. N. Engl. J. Med. 2019 Jul 11; 381(2): 111–120. PubMed Abstract | Publisher Full Text\n\nOSF: Underlying data for ‘Sense of personal agency towards mitigating the threat of antibiotic resistance: a focus group study with parents of children under 5 years old, conducted mid-pandemic’.Publisher Full Text"
}
|
[
{
"id": "240493",
"date": "01 Mar 2024",
"name": "Ateya Megahed Ibrahim",
"expertise": [
"Reviewer Expertise nursing"
],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\n1. The study's sample size seems quite small, particularly considering the diversity of perspectives that could exist among parents of children under 5. Have you considered expanding the sample to capture a wider range of experiences and attitudes?\n2. While the study delves into parents' knowledge, attitudes, and behaviors regarding antibiotic use and AMR, it may benefit from a deeper exploration of the socio-economic factors influencing these attitudes. How do income levels, access to healthcare, and education impact parents' decisions regarding antibiotic use for their children?\n3. The focus groups were conducted mid-pandemic, which undoubtedly influenced participants' perspectives. However, the study could provide a more thorough analysis of how specific aspects of the pandemic (e.g., lockdowns, fear of COVID-19 transmission) shaped parents' views on antibiotic use and infection prevention.\n4. It's interesting that the study primarily focused on mothers from parenting networks. Given that caregiving responsibilities are often shared among parents, did the exclusion of fathers limit the breadth of perspectives represented in the study?\n5. The study mentions the Framework Method of analysis but lacks detail on how themes were identified and analyzed within this framework. Providing more transparency on the analytical process would strengthen the study's methodological rigor.\n6. While the study acknowledges the influence of socio-cultural background on parents' KABs, it could benefit from a deeper exploration of how cultural beliefs and practices intersect with attitudes towards antibiotic use and AMR. How do cultural perceptions of illness and healthcare providers shape parents' decisions in this context?\n7. The study highlights the need to engage parents in discussions about appropriate antibiotic use. However, it's unclear how these findings can be translated into actionable strategies for healthcare providers and policymakers. What specific interventions or educational initiatives could effectively address the gap between knowledge and behavior identified in the study?\n8. The study emphasizes the importance of individual agency in mitigating AMR. However, it could delve deeper into the structural barriers that may hinder parents' ability to make informed choices about antibiotic use, such as limited access to healthcare resources or systemic inequalities in healthcare delivery.\n9. The discussion around the pandemic's influence on parents' views lacks nuance. How did factors like media messaging, government guidelines, and personal experiences with COVID-19 shape parents' perceptions of infection prevention and antibiotic use?\n10. As the lead researcher is a mother of young children and a medical journalist, there may be inherent biases that influence the study's design and interpretation of results. How did the research team mitigate potential biases and ensure objectivity throughout the study process?\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": "360546",
"date": "06 Feb 2025",
"name": "Huong Vu",
"expertise": [
"Reviewer Expertise Antimicrobial stewardship",
"antimicrobial resistance",
"implementation science"
],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe study examines KABs in relation to antibiotic use and AMR among parents of children <5 y.o. and identifies the relevance of COVID-19 pandemic experience and impact in the issue of AMR, using qualitative research. The manuscript can be improved in the following aspects:\nThe suggestion to draw on the experience of vaccination and mitigation measures during COVID-19 pandemic to the issue to AMR is relevant. However, the authors need to discuss/ elaborate on further the potential challenges associated with the AMR issue in comparison with COVID-19. Particularly, AMR is a silent pandemic – unlike COVID-19, and the consequences of AMR on society appear to take a much longer time to occur. In addition, did the authors directly ask the participants if/how the lessons learnt from COVID-19 control could be applied to AMR? There seems to be a lack of direct reference to the participants’ discussion quoted in the manuscript. If this was not asked directly to the participants, but inferred from the authors’ point of view – this should be made clear. Introduction: 2nd paragraph - “antibiotic resistance” should be “AMR” for consistency throughout? 4th paragraph – mentioning the “2022 data showing wide variation across practices”, please summarize what are the variations. Introduction: Last paragraph – mentioning “This study aimed to obtain a snapshot of the perceptions and behaviours…”, here the authors mentioned “perceptions” instead of “knowledge, attitudes” as described in the rest of the document. Why such a difference? What were considered “perceptions” as compared to “knowledge, attitudes” in this study? Methods: please indicate how many participants were recruited through snowball sampling and how this can influence the data collected; can snowball sampling generate a sample size with more similar participants with similar viewpoints and experience? Methods: the authors mentioned “piloting by two parents”, who were they? Were they included in the study sample size? Were they included in the protocol approved by ethics committee? Data analysis: the authors mentioned about “alternative open access software to NVivo exist for example, Taguette or RDQA”, did the authors also use these alternatives and what were the added values? Data analysis: “data saturation point” was mentioned; but how this was determined? What did the authors do when “no new codes were generated”? Did this mean the sample size (individual participants and groups) was somehow influenced when the saturation point was reached? The sentence was not clear. Results: Please specify clearly if the 2 participants dropped out and 1 other- “a medical doctor”, “conflicted”- included in the total sample size of 14 parents participating the 3 groups. Table 3: The subthemes were sometimes not clear, it would be better if each of the subthemes were made with a clear meaning. I understand that this could be due to the fact that each subtheme was a summary of the content but in some cases these were not clear what the authors wanted to summarize specifically. Eg. “specific or broad spectrum”, “cultural shift (or not)”, “More pressing problems in the world”, “misuse – overuse”..., these were not clear, and some undefined words such as “ABX”. Results: Use of the words such as “sometimes”, “some”, “most”, “relatively common”… should be more specific by specifying how many of the participants stating a particular result. There were only 14 participants, which makes it less convincing when using the above words when describing the results. Discussion – 1st sentence is not clear, and the reference of the 2016’s review is no longer updated, suggest to rewrite and use more updated references.\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? Yes",
"responses": []
}
] | 1
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https://f1000research.com/articles/11-1487
|
https://f1000research.com/articles/11-1236/v1
|
31 Oct 22
|
{
"type": "Review",
"title": "Circadian stabilization loop: the regulatory hub and therapeutic target promoting circadian resilience and physiological health",
"authors": [
"Eunju Kim",
"Seung-Hee Yoo",
"Zheng Chen",
"Eunju Kim",
"Seung-Hee Yoo"
],
"abstract": "The circadian clock is a fundamental biological mechanism that orchestrates essential cellular and physiological processes to optimize fitness and health. The basic functional unit is the cell-autonomous oscillator, consisting of intersecting negative feedback loops. Whereas the core loop is primarily responsible for rhythm generation, auxiliary loops, most notably the secondary or stabilization loop, play pivotal roles to confer temporal precision and molecular robustness. The stabilization loop contains opposing nuclear receptor subfamilies REV-ERBs and retinoic acid receptor-related orphan receptors (RORs), competing to modulate rhythmic expression of the basic helix-loop-helix ARNT like 1 (Bmal1) genes in the core loop as well as other clock-controlled genes. Therefore, REV-ERBs and RORs are strategically located to interface the oscillator and the global transcriptomic network, promoting cellular homeostasis and physiological fitness throughout lifespan. Disruption of REV-ERB and ROR functions has been linked with diseases and aging, and pharmacological manipulation of these factors has shown promise in various mouse disease models. Nobiletin is a natural compound that directly binds to and activates RORα/γ, modulating circadian rhythms, and shows robust in vivo efficacies to combat clock-associated pathophysiologies and age-related decline. Results from several studies demonstrate an inverse relation between nobiletin efficacy and clock functional state, where nobiletin elicits little effect in young and healthy mice with growing efficacy as the clock is perturbed by environmental and genetic challenges. This mode of action is consistent with the function of the stabilization loop to promote circadian and physiological resilience. Future studies should further investigate the function and mechanism of REV-ERBs and RORs, and test strategies targeting these factors against disease and aging.",
"keywords": [
"Circadian oscillator",
"core loop and stabilization/secondary loop",
"REV-ERBs and RORs",
"ligands and drugs",
"circadian amplitude and resilience",
"physiological health",
"healthy aging"
],
"content": "The circadian timing system and health implications\n\nCircadian rhythms are daily cycles of intrinsic processes in living organisms. While light/dark cycles of our environment are the predominant input (or zeitgeber, time giver) to reset our internal rhythms, it is now clear that other factors including feeding-fasting state, nutrients, physical activity, and temperature are all capable of manipulating the circadian cycle.1–3 Fundamentally, the circadian timing system is a molecular circuit governing cellular and physiological homeostasis throughout lifespan. Alterations to this clock machinery, by either environmental stresses or genetic defects, have been shown to cause or correlate with dysfunction of diverse physiological processes and increased risks for various diseases involving both peripheral organs and the brain.4–6\n\nAt the pinnacle of the circadian timing system is the master pacemaker located in the suprachiasmatic nucleus (SCN) of the hypothalamus. The SCN clock synchronizes semi-autonomous cellular oscillators in other brain regions and peripheral organs through neuronal and hormonal signals.7–9 The ubiquitous cellular oscillator, present in the SCN and throughout the body, contains interlocked transcriptional and translational feedback loops controlling the expression of downstream target genes.10 The core clock genes functioning in the oscillator include circadian locomotor output cycles kaput (Clock)/neuronal PAS domain-containing protein 2 (Npas2), basic helix-loop-helix ARNT like 1 (Bmal1), period 1 (Per1)/period 2 (Per2)/period 3 (Per3), cryptochrome 1 (Cry1)/cryptochrome 2 (Cry2), Rev-erba/Rev-erbb (nuclear receptor subfamily 1 group D member 1/2 (Nr1d1)/2)), and retinoic acid receptor-related orphan receptor alpha (Rora)/retinoic acid receptor-related orphan receptor beta (Rorb)/Retinoic acid receptor-related orphan receptor gamma (Rorc) (Nr1f1/2/3). By acting on consensus promoter elements or directing the expression of secondary regulators of gene expression, the encoded core clock proteins play a prevalent role in the global gene expression landscape where more than 80% of genes have been shown to oscillate in at least one location in the body.11,12\n\nPerhaps one of the most important physiological functions of the clock is to safeguard energy homeostasis.13,14 It has been postulated that an evolutionary origin of the circadian system is energy partitioning: photosynthesis using oxygen during the day and anaerobic metabolism including nitrogen fixation at night.15 In mammals, central and peripheral clocks coordinately drive rhythmic expressions of metabolic-related genes in organs with high metabolic activity including liver, muscle, and adipose tissue.3,16–18 Over the past 15 years or so, a growing body of evidence has established that the clock gene machinery influences energy homeostasis directly and genetic mutations in clock genes lead to metabolic dysfunctions, including deficient insulin resistance, glucose intolerance, leptin resistance, and abnormal glucocorticoid and melatonin levels.17,19,20 In accordance, human subjects who were exposed to a controlled circadian misalignment condition displayed glucose intolerance, insulin resistance, and other comorbidities.21–23 In addition, our lifestyle choices that affect circadian rhythms may also evoke adverse metabolic consequences. For example, external stimuli including abnormal light exposure,24 jet-lag,20,25 and high-fat diet induced-obesity26,27 can trigger desynchronization of the internal clock accompanied by many tissue disorders. Furthermore, sleep deprivation, a common occurrence in modern lifestyle, is associated with increased body mass index and type 2 diabetes incidence and has been identified as an independent risk factor for hypertension, obesity, and coronary heart disease.28,29 In addition, sleep and feeding alterations and shift work are highly correlated with elevated metabolic syndrome markers such as triglycerides, and lower high-density lipoprotein (HDL)-cholesterol levels.29–31\n\nDysregulated clocks are also involved in brain dysfunction and diseases.32–34 Sleep is well known to be regulated by the clock, and elegant studies combining human genetics and mechanistic investigation have revealed molecular links between several mutations in clock genes, including PER2 and casein kinase I isoform delta (CSNK1D), and sleep disorders.35 An emerging area of interest is the crosstalk between the clock and neurodegenerative diseases.36–38 Circadian clocks have been shown to control several aspects of brain functions linked to neurodegeneration including dopamine synthesis, inflammatory response, oxidative stress, and cellular metabolism.33,39 Consistently, circadian and sleep disruptions are closely associated with neurodegenerative diseases including Alzheimer’s disease and Parkinson’s disease,40 as evidenced by amyloid-beta (Aβ) oscillation,41 sundowning behaviors,42 and neuronal inflammation in mouse genetic mutants.40\n\nGiven the fundamental role of the clock in cellular and physiological homeostasis and the myriads of chronic diseases associated with circadian dysregulation, it is not surprising that age-related decline over time is strongly correlated with and likely exacerbated by dysfunction in the clock system.43 It is well known that a number of physiological parameters display blunted circadian rhythms during aging, including sleep, temperature, and hormone secretion.43,44 More recently, global transcriptomic profiling revealed profound rewiring in the clock network, notably dampening of oscillatory gene expression in accordance with the physiological decline.45 A key role of the circadian rhythms in aging is further highlighted by two large-scale gene profiling studies where circadian gene expression changes emerged from unbiased analyses as a top underlying pathway during aging.46,47 For example, a comparative multi-tissue gene profiling approach was undertaken to search for pathways correlated with maximum lifespan in 26 species, and identified the circadian system as a pillar that governs metabolic and inflammatory pathways for longevity regulation.47 Furthermore, interventional fasting paradigms designed to incorporate circadian timing were recently reported to markedly prolong lifespan in Drosophila and mice,48,49 including 35% lifespan extension in male mice. The convergent spotlight on circadian remodeling during aging provides compelling evidence for the notion that a robust circadian system is key to health and healthspan.50\n\n\nThe stabilization loop\n\nThe core loop of the oscillator is primarily responsible for generating the near-24hr rhythm via the negative feedback between CLOCK/BMAL1 and CRY/PER. Through binding to E-box elements, the CLOCK/BMAL1 heterodimer activates the expression of many Clock-Controlled Genes (CCGs). As PER/CRY proteins accumulate and reach critical levels in the cytoplasm, they translocate to the nucleus to inhibit the activity of CLOCK/BMAL1, thereby inhibiting their own transcription.10 On the other hand, the secondary loop, mainly involving the opposing transcription factors REV-ERBs and RORs,51,52 confers stability and robustness for the core loop, and is also strategically located at the interface between the core oscillator and many downstream clock-controlled genes (Figure 1). Growing evidence suggests a regulatory and therapeutic potential of the stabilization loop in physiology, disease, and aging.53\n\nThe core oscillator consists of core and stabilization loops as indicated by the dotted circle. The REV-ERB and retinoic acid receptor-related orphan receptors (RORs) compete at the consensus ROR response elements (ROREs) of target gene promoters, including basic helix-loop-helix ARNT like 1 (Bmal1) and many clock-controlled genes (CCGs), to regulate circadian transcription in a tissue-specific manner. They may interact via other mechanisms and in clock-independent processes – see text for details. REV-ERBs and RORs play regulatory roles in many tissue and organismal functions and targeting these receptors by small-molecule agents may strengthen circadian resilience, ultimately conferring beneficial effects to promote health and health span. CLOCK, circadian locomotor output cycles kaput; CRY, cryptochrome; PER, period.\n\nREV-ERBs and RORs, the main components of the stabilization loop, are multi-functional nuclear receptors to repress and activate target gene expression, respectively.52,54,55 REV-ERBs and RORs bind as monomers to the same consensus sequence, namely the ROR response elements (ROREs, or RREs) consisting of an (A/G) GGTCA flanked by an A/T-rich 5’ in the regulatory regions of the target genes.56–58 In the core oscillator, perhaps the most important function of REV-ERB/ROR is to maintain and fine-tune the oscillatory expression of Bmal1, encoding the chief circadian transcription activator in the core loop, thereby serving as a stabilizing mechanism.52,55,59,60 In addition, several other core clock genes have been shown to possess the RORE elements in their promoter region,61 suggesting an important stabilizing function by these opposing regulators. More broadly, gene expression and profiling studies have illustrated a prominent role of REV-ERBs and RORs in transcriptomic landscape in various tissues/cells.62–66 For example, studies of liver-specific RORα/RORγ double knockout mice revealed a key role of RORs in lipogenesis, via direct transcriptional regulation of the insulin-induced gene 2 (Insig2)-sterol regulatory element-binding protein 1 (SREBP) cascade.66\n\nTraditionally REV-ERBs and RORs are considered to interact and compete as repressors and activators on shared target genes. While Ror expressions display relatively moderate circadian oscillatory amplitude, Rev-erbs are among the most oscillatory genes (highest amplitude) in both protein and mRNA expression.65,67 It is believed that their opposing functions and circadian patterns (expression and promoter recruitment) play a key role in amplitude regulation of clock-controlled genes. In a pioneering study, the mRNA oscillation of Bmal1, Clock, and Cry1 genes show strong amplitude in wild-type animals but is diminished in Rev-erbα deficient mice.55 Although dispensable for Bmal1 oscillation per se, RORs were found to contribute to its amplitude.52,68,69 More recently, correlation analysis of publicly available mouse circadian transcriptomic datasets, including both microarray and RNA-sequencing, identified a strong correlation between Rorc expression level and amplitude with the percentage of cycling transcripts in respective tissues, consistent with a regulatory role of RORγ in circadian oscillation.70 In addition to jointly regulating Bmal1 transcription, additional modes of genetic and molecular interplay exist between REV-ERBs and RORs. Rorc itself contains a functional RORE element in its promoter, therefore subject to transcriptional regulation by REV-ERBs/RORs.61 Moreover, molecular studies have demonstrated a facilitated recruitment mechanism where REV-ERBs are recruited to the target gene promoter by RORs, in a process that requires chromatin remodeling by SWI/SNF factors.71 These observations suggest an interconnectivity, rather than a simple competition, relationship between these master regulators. As discussed below, our studies of an ROR agonist, nobiletin (NOB), provide further evidence that RORs, and likely REV-ERBs, regulate circadian gene expression levels and amplitude in a context-dependent manner, potentially dictated by an inherent requirement to maintain circadian and physiological resilience.\n\nConsistent with the broad gene regulatory roles of REV-ERBs and RORs, mouse genetic mutants exhibit various circadian and physiological phenotypes. Rev-erba (Nr1d1)-deficient mice showed disrupted circadian rhythms including a shorter period length (0.5 h) and exaggerated light-induced phase shifts compared to wild-type (WT) mice.55,72 Rev-erbb (Nr1d2) knockout (KO) mice also displayed a strong diurnal change of gene expression including inhibition of Bmal1 transcription.51 While individual KO mice retained circadian rhythmicity, Nr1d1/2 double knockout led to severe disruption of overt rhythms, consistent with functional redundancy between these two subtypes.68,73,74 Rora- and Rorb-deficient mice were reported to display altered circadian behavior such as circadian locomotor activity and shortened period length, while no significant alteration in wheel activity is apparent in Rorc-deficient mice.52,68,69,75,76 These results indicated that REV-ERBs and RORs are required for the maintenance of normal circadian behavior and period length.\n\nWith respect to tissue physiology, REV-ERBs and RORs show overlapping but distinct expression patterns and their deficiency led to a wide range of other physiological deficits. REV-ERBα and REV-ERBβ are expressed in skeletal muscle, adipose tissue, liver, and brain with tissue-specific patterns. Whereas REV-ERBα is broadly expressed in a rhythmic manner in many tissue types with robust amplitude, REV-ERBβ is highly expressed in fewer tissues including certain brain regions (pineal and prefrontal cortex), thyroid, uterus, and pituitary. Deficiency of both Rev-erbs causes liver steatosis, in contrast to relatively minor changes upon loss of each subtype alone.73,77\n\nLike REV-ERBs, the three members of the ROR subfamily, RORα, RORβ, and RORγ, display significant sequence similarities. RORα is expressed broadly, notably in skeletal muscle, liver, kidney, lungs, adipose tissue, skin, and brain.62 Rora KO (Rora−/−) and staggerer mutant (Rorasg/sg) mice displayed debilitating cerebellar ataxia and are mostly infertile.52,69,78,79 RORα-deficient mice also showed a multitude of other defects including thin long bones80 and abnormal retinal development,81,82 the latter corresponding to high expression levels of RORα in the ganglion cell layer, the inner nuclear layer, and cone photoreceptors in the outer layer. RORβ expression is more limited, mainly in the nerve system. Rorb−/− mice exhibited reproductive abnormality and serious degeneration of postnatal retina.59 RORγ is expressed in several peripheral tissues including skeletal muscle, liver, kidney, adipose tissue, and particularly thymus.62 In accordance, RORγ KO led to reduced levels of thymocytes and abnormal lymphoid organ development.83 This and many other studies have since established a key role of RORyt as the master transcription factor for Th17 cell development, although circadian clock involvement in this function is not fully understood. Several studies have also examined double disruption of RORs, providing evidence for their overlapping functions. As mentioned above, in the liver where both RORα and RORγ are expressed, double Rora/c KO led to strong disruption of lipogenesis via a direct regulation of the Insig2 gene.66\n\n\nTherapeutic relevance of REV-ERBs and RORs\n\nREV-ERBα and RORs have been implicated in various diseases including metabolic diseases, immune diseases, and cancers.53,62,84,85 REV-ERBα and RORs expression show altered expression and disrupted rhythm during disease development.86–88 Furthermore, alteration of REV-ERBα and RORs affects the organism susceptibility to diseases in both humans and mice and is involved in many pathways associated with pathological processes and diseases.62,85,88,89–91\n\nMyriad studies have illustrated a regulatory role of REV-ERBs and RORs in energy metabolism. REV-ERBα was found to regulate de novo glucose synthesis.92–94 In accordance, REV-ERBα-deficient mice showed a higher plasma glucose level,73,95 whereas activation of REV-ERBα diminished plasma glucose levels, improving disease phenotypes.92–94,95 RORs are also involved in glucose metabolism. Single nucleotide polymorphism in RORA (rs7164773) has been shown to correlate with increased risk of type 2 diabetes in the Mexico Mestizo population, providing human genetic evidence for a role of RORα in insulin sensitivity.96 In addition, it was reported that RORα was required for the secretion of FGF21, a hormone associated with glucose tolerance and hepatic lipid metabolism.97–99 In another study, RORγ was found to regulate transcription of various genes involved in glucose metabolism including glucose-6-phosphatase (G6p), phosphoenolpyruvate carboxykinase 1 (Pck), and glucose transporter 2 (Glut2), and Rorc-deficient mice in fact displayed a significantly higher insulin sensitivity and glucose tolerance than WT mice, particularly at ZT4–6.100 In pharmacological studies, SR1078, an agonist of RORα and RORγ, was able to improve insulin sensitivity, blood glucose, and triglyceride levels in diabetic rodents.101 In comparison, mice treated with SR3335, an RORα inverse agonist, showed dramatically decreased glucose levels in the plasma compared with the control mice, by inhibiting Pck expression and gluconeogenesis.102\n\nWith respect to lipid metabolism, Rev-erba−/− mice showed increased very low-density lipoprotein (VLDL) and triglyceride levels, consistent with the observed upregulation of apolipoprotein C-III (Apoc3), a critical regulator in triglyceride metabolism.103,104 Depletion of both Rev-erbs in the liver synergistically de-repressed several metabolic genes as well as genes that control the positive limb of the molecular clock.105 Consistent with these genetic results, administration of the REV-ERB agonist SR9009 decreased cholesterol levels in the plasma in both wild-type and low-density lipoprotein receptor (Ldlr) null mice through downregulating cholesterol biosynthesis gene expression.106 Extensive mouse studies also point to a key role of RORs in lipid metabolism. In Rorαsg/sg staggerer mice, expression levels of hepatic sterol regulatory element-binding protein 1, isoform c (Srebp-1c), and fatty acid synthase (Fas) were decreased, whereas expression of PGC-1α and β, coactivators involved in oxidative metabolism and gluconeogenesis, were elevated.64,107 At the molecular level, gene expression and cistrome analysis showed that RORα and/or RORγ broadly regulate genes involved in lipid metabolism in both liver and muscle tissues.66,100,108,109 Furthermore, structural and biochemical studies identified lipid moieties, mainly cholesterol metabolic intermediates, as possible endogenous ligands for RORα/γ, consistent with the notion that RORs may function as a lipid sensor in the regulation of lipid metabolism.64,107,108,110–113\n\nMounting evidence indicates circadian rhythms in immunity and inflammation. For example, rheumatoid arthritis patients exhibit diurnal variations in functional disability such as joint pain and stiffness in morning time.114,115 REV-ERBα deletion abolished the diurnal rhythms of various inflammatory factors and aggravates inflammation in diseases including autoimmune encephalomyelitis,116,117 fulminant hepatitis,89 neuroinflammation,118,119 heart failure,120,121 myocardial infarction,122 and ulcerative colitis.116,123 At the molecular level, REV-ERBα regulates rhythmic transcription of inflammation-related genes involved in macrophage polarization, immune cell differentiation, and NF-κB signaling.88,123 For example, REV-ERBα was found to obstruct NF-κB signaling in human endometrial stroma cells and macrophages/microglia cells in mouse models, suppressing expression of inflammatory genes such as IL-1β, IL-6, IL-18, tumor necrosis factor alpha (Tnfα), NACHT, LRR and PYD domains-containing protein 3 (Nlrp3), and C-C motif chemokine 2 (Ccl2).88,118,119,124 Activation of REV-ERBα by SR6472 inhibits NF-κB signaling and NLRP3 inflammasome activity to prevent cytokines and chemokines productions, consistent with an anti-inflammatory role of REV-ERBα.2,88,119\n\nRORs also play important roles in immunity.85 Extensive research has established RORγt, a subtype of RORγ, as a master regulator of Th17 cell differentiation and therefore highly involved in autoimmune diseases.125 In Th17 cells, RORγt is expressed at dramatically higher levels during daytime than at nighttime.126 This diurnal expression pattern in turn up-regulates BMAL1-dependent Rev-erb expression during daytime and conversely represses NFIL3 transcription. Given the central role of RORγt in Th17 cells, several compounds targeting RORγt have been tested in autoimmune disease models. For example, SR1001, an RORα and RORγ inverse agonist, inhibited Th17 cell differentiation under autoimmune disease conditions.127 Moreover, this effect is associated with decreased expression of several cytokines such as IL17A, IL17F, IL21, and IL22 by specially targeting TH17.128,129 Likewise, SR2211, an RORγ inverse agonist, suppressed Th17 cell differentiation and reduced IL17a and IL23R expression levels as well as intracellular IL17 protein level.130\n\nCircadian disruption can adversely impact brain development and function, potentially leading to various mood and neurological disorders.33,38 Previously, Rev-erbb knockout mice were found to exhibit enhanced anxiety, and treatment of an REV-ERB agonist showed anxiolytic effects.131 On the other hand, acute administration of SR8278, a REV-ERB antagonist, improves anxiety symptom and maniac-like behavior.132,133 Furthermore, REV-ERBα was shown to diminish fatty acid-binding protein 7 (Fabp7) expression, thereby impairing neuronal differentiation and depleting neuronal progenitor cells.134,135 Relatedly, deficiency of REV-ERBα adversely affected hippocampal neurogenesis, which contributes to altered mood behaviors.136\n\nRORα is highly expressed in several brain regions such as cerebellar Purkinje cells (PC) and thalamus, and functions to regulate brain development.62,137–139 The classical RORα-deficient staggerer mice have been shown to present severe ataxia because of cerebellar neurodegeneration78,79,140 and abnormal PC development.78,140,141 Likewise, Rora KO mice exhibit reduced numbers and sizes of PC in the cerebellar region reminiscent of clinical observations from patients with autism-spectrum disorder (ASD).142,143 RORα also showed neuroprotective effects in astrocytes and neurons during hypoxia.110,144 RORβ is highly expressed in the retina, pineal gland, and suprachiasmatic nucleus, and has been implicated in visual function, motor ability, and circadian rhythms.62,76,145 For example, RORβ-deficient mice showed abnormal motor and olfactory functions, anxiety control, and alterations in circadian behavior.75 The noteworthy question regarding a potential functional overlap in the neuronal system between RORα and RORβ remains to be investigated.\n\nREV-ERBs (α and β) and RORs (α and γ) are highly expressed in the skeletal muscle where they modulate myofiber types and energy metabolism62,146 and may be targeted against myopathies.147 In an early study, REV-ERBα-deficient mice showed a marked increase in the relative amount of the slow (type I) myosin heavy chain (MyHC) isoform compared to WT controls.146 Extensive research since has further revealed the regulatory roles of REV-ERBs in skeletal muscle function.84,148,149 For example, REV-ERBβ has been implicated in skeletal muscle lipid metabolism since ectopic expression of its dominant-negative form upregulated expression of genes associated with fatty acid uptake in skeletal muscle.150 Consistently, SR8278, an antagonist of REV-ERBs, was found to activate expression of myogenesis genes including Myogenic determination 1 (Myod), Myogenin (Myog), and Major histocompatibility complex 3 (Mhc3), suggesting a role of REV-ERBs in myogenesis.151\n\nLoss-of-function studies also suggest an important role of RORα in skeletal muscle metabolism.152 For example, ectopic expression of a dominant-negative RORα in C2C12 cells or mouse skeletal muscle broadly alters the expression of genes associated with lipid metabolism, lipogenesis, and energy expenditure, including carnitine palmitoyltransferase-1 (Cpt1), caveolin 3 (Cav3), and Srebp1c and its downstream targets.108,153\n\nREV-ERBα has been implicated in the progression and development of various cancers.154 Activation of REV-ERBα by SR9009 and SR9011 was found to confer cancer cell-selective cytotoxicity as well as in vivo efficacy against glioma, and autophagy and lipogenesis were identified as cellular hallmarks closely associated with this anti-cancer activity.155 In a recent study investigating lung adenocarcinoma-associated cachexia,156 REV-ERBα functions as a key effector whose exaggerated turnover contributes to gluconeogenesis gene induction and glucose production in mice.\n\nA number of studies have shown that RORα expression is significantly decreased during tumor development and progression, and exogenous RORα expression repressed cell proliferation and tumor growth.157–162 For example, down-regulated RORα expression has been observed in colorectal cancer and mammary cancer, and is associated with poor prognosis in patients with hepatocellular and breast carcinoma.157,158,160,161,163,164 Conversely, restoring RORα expression suppressed cell migration and tumor growth of breast cancer cells as well as metastasis in nude mice, which was accompanied by up-regulated expression of semaphorin 3F (SEMA3F), a tumor suppressor that reduces tumor growth and invasion.160 In colon cancer HCT116 cells treated with DNA-damage agents, a p53-RORα crosstalk was required for apoptosis, where Rora gene transcription was dependent on p53 and RORα in turn rendered greater p53 protein stability.165 In RORγ deficient mice, there was an aggravated development of T-cell lymphomas within the first months after birth, which rapidly metastasized to other organs including liver and spleen.166\n\n\nNobiletin (NOB): A natural ROR agonist\n\nNOB is a natural bioactive polymethxylated flavonoid.167,168 Many studies have provided functional evidence both in vitro and in vivo for its biological efficacy in diverse disease models,167,169,170 including metabolic diseases and inflammation. In our previous unbiased chemical screen, we identified NOB, along with its close analog tangeretin, as a clock-enhancing small molecule in cell-based circadian reporter assays.171 Focusing on NOB, we demonstrated a circadian clock-dependent efficacy to blunt obesity and metabolic dysfunction in mouse models, and importantly identified RORα and RORγ as its direct targets via radioactive ligand binding assays. Following this seminal discovery, a number of published studies, from our group and others, provide further functional evidence that NOB plays a beneficial role in strengthening circadian physiologies in various mouse models, including aging, metabolic disorders, cardiovascular disease, and Alzheimer’s disease (AD).170,172–180 In further support of NOB as an anti-inflammatory agent, recent studies demonstrated a potent role of NOB against neuroinflammation and astrogliosis, accompanied by mitigation of Aβ plaque deposition, in an amyloid AD mouse model.181 Given the increasing appreciation of circadian rhythms in aging, recent studies have also tested its effect in aging models. In naturally aged mice fed with either regular or high-fat diets (HFD), NOB was found to promote healthy aging at several levels, including metabolic homeostasis, inflammatory markers, tissue functions, and systemic behaviors.173 An important target organ is skeletal muscle, where circadian gene reprogramming and metabolomic alteration support an improved mitochondrial function, accompanied by respiratory supercomplex formation. Notably, while NOB-mediated improvement in general healthy aging parameters seems more pronounced in metabolically challenged aged mice (HFD fed) than in those fed with regular diet, the latter group showed an extension of median lifespan, but not maximum lifespan.173 In comparison, NOB was found to exhibit longevity effects in C. elegans, extending median lifespan by up to 21%.182 Overall, these and many other studies underscore a strong health-promoting effect of NOB, at least in part via circadian mechanisms.\n\nMechanistic studies have begun to shed light on the circadian modulatory action of NOB. In addition to its clock amplitude-enhancing effects, NOB also alters the other two cardinal circadian parameters, period, and phase, at least in vitro.171,180 Following chronic treatment in vivo (10-12 weeks), NOB was able to strengthen oscillatory amplitude, as well as peak expression, of core clock components at both transcript and protein levels in HFD-fed mice, and wheel-running activity was also increased at nighttime.171 Given the extensive crosstalk between clocks and metabolism/physiology, these overt enhancements of circadian rhythms may result from both direct and indirect effects of NOB on the core oscillator/RORs and clock-regulated downstream functions, respectively. Acute in vivo effects on circadian rhythms remain to be investigated. Another important issue is related to the varying effects of NOB according to the clock functional state. There seems to be a general inverse correlation between NOB efficacy and clock health. For example, in young and healthy mice under normal husbandry conditions, NOB showed essentially no effects on circadian and metabolic functions, contrary to the profound improvements in obese or diabetic mouse models.171,176,177 Likewise, as mentioned above, aged mice further challenged with HFD known to dampen circadian rhythms showed a greater responsiveness to NOB in healthy aging compared with aged mice fed with normal diets.173 A similar pattern was observed between WT and AD mice at old ages (>22 months) where NOB was found to mitigate neuroinflammation more markedly in the latter, correlating with a more severe circadian disruption in AD mice.181 These in vivo results together suggest a role of NOB to enhance circadian resilience toward restoring normal circadian rhythms that may have evolved to operate within a physiological range. Either dampening or indiscriminately enhancing the normal circadian rhythms is likely detrimental to organismal health.\n\nFurther research should investigate the downstream cellular mechanisms intersecting with the clock machinery. In a recent study, an inhibitory function of NOB against triple-negative breast cancer (TNBC) was found after cell line screening.183 Both in vitro and in xenografts, NOB was able to blunt TNBC cell growth, either alone or in combination with chemotherapeutic agents. The cellular mechanism entailed, at least in part, suppression of NF-κB signaling, via a pathway where activation of RORs by NOB increased expression of its downstream target gene encoding IκBα, and ChIP analysis showed that ROR recruitment to the IκBα gene promoter was potentiated.183 While this study illustrates a cellular pathway targeted by NOB in TNBC, it should be noted that the TNBC cells examined do not have a functional clock despite detectable clock gene expression, and NOB was not able to restore the core oscillator in these cells.183,184 Therefore, this is a scenario that NOB effects are mediated by ROR transcriptional regulation independent of oscillator function. However, since the host mice have circadian rhythms, whether NOB modulates host rhythms as part of the effect against TNBC remains to be investigated.\n\n\nConcluding remarks\n\nAccumulating evidence from molecular, genetic and interventional studies highlight a critical role of the circadian secondary/stabilization loop, specifically the REV-ERBα/β and RORα/β/γ nuclear receptors, in linking the core oscillator with physiology and behavior under both normal and pathological conditions. These are multi-functional transcription factors, playing important regulatory roles in circadian regulation as well as other processes not primarily tied to the clock (e.g., RORγt in Th17 differentiation and autoimmunity). It is therefore a challenge to dissect the underlying mechanisms and devise disease-specific interventions from the circadian perspective.147 Whenever possible, detailed circadian characterization should be performed, especially at the tissue and organismal levels. As illustrated by pharmacological studies targeting these factors,53,185 including those on NOB, the concept of circadian resilience, or restoration of homeostatic clock function, should be an important consideration regarding intervention. Finally, given the tissue-specific nature of circadian regulation and the growing evidence for inter-organ communication with the clock system,186 the functional effects, mechanistic pathways and interventional approaches should be interrogated accordingly in an integrative manner. In that regard, distribution and functional redundancy among the subtypes of these receptors should be considered. Despite the inherent complexity and practical challenges, targeting the circadian machinery, including the secondary loop, represents an exciting frontier in the 4th dimension for research and medicine.\n\n\nAuthor contributions\n\nConceptualization: Z.C.; Original draft preparation: E.K. and Z.C.; Review and Editing: S.-H.Y. and Z.C.",
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Dev. 1998; 70: 147–153. PubMed Abstract | Publisher Full Text\n\nChen XR, et al.: Mature Purkinje cells require the retinoic acid-related orphan receptor-α (RORα) to maintain climbing fiber mono-innervation and other adult characteristics. J. Neurosci. 2013; 33: 9546–9562. PubMed Abstract | Publisher Full Text\n\nDoulazmi M, et al.: A comparative study of Purkinje cells in two RORα gene mutant mice: staggerer and RORα−/−. Dev. Brain Res. 2001; 127: 165–174. PubMed Abstract | Publisher Full Text\n\nJanmaat S, et al.: Age-related Purkinje cell death is steroid dependent: RORα haplo-insufficiency impairs plasma and cerebellar steroids and Purkinje cell survival. Age. 2011; 33: 565–578. PubMed Abstract | Publisher Full Text\n\nJolly S, et al.: Cell-autonomous and non-cell-autonomous neuroprotective functions of RORα in neurons and astrocytes during hypoxia. J. Neurosci. 2011; 31: 14314–14323. PubMed Abstract | Publisher Full Text\n\nSrinivas M, Ng L, Liu H, et al.: Activation of the blue opsin gene in cone photoreceptor development by retinoid-related orphan receptor β. Mol. Endocrinol. 2006; 20: 1728–1741. PubMed Abstract | Publisher Full Text\n\nPircher P, Chomez P, Yu F, et al.: Aberrant expression of myosin isoforms in skeletal muscles from mice lacking the rev-erbAα orphan receptor gene. Am. J. Phys. Regul. Integr. Comp. Phys. 2005; 288: R482–R490. PubMed Abstract | Publisher Full Text\n\nWelch RD, Flaveny CA: REV-ERB and ROR: therapeutic targets for treating myopathies. Phys. Biol. 2017; 14: 045002. PubMed Abstract | Publisher Full Text\n\nXiong X, Gao H, Lin Y, et al.: Inhibition of Rev-erbalpha ameliorates muscular dystrophy. Exp. Cell Res. 2021; 406: 112766. PubMed Abstract | Publisher Full Text\n\nBoulinguiez A, et al.: NR1D1 controls skeletal muscle calcium homeostasis through myoregulin repression. JCI Insight. 2022; 7. PubMed Abstract | Publisher Full Text\n\nRamakrishnan SN, Lau P, Burke LJ, et al.: Rev-erbβ regulates the expression of genes involved in lipid absorption in skeletal muscle cells: evidence for cross-talk between orphan nuclear receptors and myokines. J. Biol. Chem. 2005; 280: 8651–8659. Publisher Full Text\n\nWelch RD, et al.: Rev-Erb co-regulates muscle regeneration via tethered interaction with the NF-Y cistrome. Mol. Metab. 2017; 6: 703–714. PubMed Abstract | Publisher Full Text\n\nFitzsimmons RL, Lau P, Muscat GE: Retinoid-related orphan receptor alpha and the regulation of lipid homeostasis. J. Steroid Biochem. Mol. Biol. 2012; 130: 159–168. PubMed Abstract | Publisher Full Text\n\nRaichur S, et al.: Identification and validation of the pathways and functions regulated by the orphan nuclear receptor, ROR alpha1, in skeletal muscle. Nucleic Acids Res. 2010; 38: 4296–4312. PubMed Abstract | Publisher Full Text\n\nErcolani L, et al.: Circadian clock: Time for novel anticancer strategies? Pharmacol. Res. 2015; 100: 288–295. Publisher Full Text\n\nSulli G, Manoogian ENC, Taub PR, et al.: Training the Circadian Clock, Clocking the Drugs, and Drugging the Clock to Prevent, Manage, and Treat Chronic Diseases. Trends Pharmacol. Sci. 2018; 39: 812–827. PubMed Abstract | Publisher Full Text\n\nVerlande A, et al.: Glucagon regulates the stability of REV-ERBalpha to modulate hepatic glucose production in a model of lung cancer-associated cachexia. Sci. Adv. 2021; 7. PubMed Abstract | Publisher Full Text\n\nZhu Y, McAvoy S, Kuhn R, et al.: RORA, a large common fragile site gene, is involved in cellular stress response. Oncogene. 2006; 25: 2901–2908. PubMed Abstract | Publisher Full Text\n\nKottorou AE, et al.: Altered expression of NFY-C and RORA in colorectal adenocarcinomas. Acta Histochem. 2012; 114: 553–561. PubMed Abstract | Publisher Full Text\n\nMoretti RM, Montagnani Marelli M, Sala A, et al.: Activation of the orphan nuclear receptor RORα counteracts the proliferative effect of fatty acids on prostate cancer cells: Crucial role of 5-lipoxygenase. Int. J. Cancer. 2004; 112: 87–93. PubMed Abstract | Publisher Full Text\n\nXiong G, Wang C, Evers BM, et al.: RORα suppresses breast tumor invasion by inducing SEMA3F expression. Cancer Res. 2012; 72: 1728–1739. PubMed Abstract | Publisher Full Text\n\nDu J, Xu R: RORα, a potential tumor suppressor and therapeutic target of breast cancer. Int. J. Mol. Sci. 2012; 13: 15755–15766. Publisher Full Text\n\nYe Y, et al.: The Genomic Landscape and Pharmacogenomic Interactions of Clock Genes in Cancer Chronotherapy. Cell systems. 2018; 6: 314–328.e2. PubMed Abstract | Publisher Full Text\n\nFu R-D, Qiu C-H, Chen H-A, et al.: Retinoic acid receptor-related receptor alpha (RORalpha) is a prognostic marker for hepatocellular carcinoma. Tumor Biol. 2014; 35: 7603–7610. PubMed Abstract | Publisher Full Text\n\nOu Z, et al.: Regulation of the human hydroxysteroid sulfotransferase (SULT2A1) by RORα and RORγ and its potential relevance to human liver diseases. Mol. Endocrinol. 2013; 27: 106–115. Publisher Full Text\n\nKim H, et al.: DNA damage-induced RORα is crucial for p53 stabilization and increased apoptosis. Mol. Cell. 2011; 44: 797–810. PubMed Abstract | Publisher Full Text\n\nUeda E, et al.: High incidence of T-cell lymphomas in mice deficient in the retinoid-related orphan receptor RORγ. Cancer Res. 2002; 62: 901–909. PubMed Abstract\n\nHuang H, et al.: The Multifunctional Effects of Nobiletin and Its Metabolites in vivo and In Vitro. Evid. Based Complement. Alternat. Med. 2016; 2016: 2918796. PubMed Abstract\n\nEvans M, Sharma P, Guthrie N: Bioavailability of Citrus Polymethoxylated Flavones and Their Biological Role in Metabolic Syndrome and Hyperlipidemia. InTech;2012; 1–19. Publisher Full Text\n\nMulvihill EE, Burke AC, Huff MW: Citrus Flavonoids as Regulators of Lipoprotein Metabolism and Atherosclerosis. Annu. Rev. Nutr. 2016; 36: 275–299. PubMed Abstract | Publisher Full Text\n\nMileykovskaya E, Yoo SH, Dowhan W, et al.: Nobiletin: Targeting the Circadian Network to Promote Bioenergetics and Healthy Aging. Biochemistry. Biokhimiia. 2020; 85: 1554–1559. PubMed Abstract | Publisher Full Text\n\nHe B, et al.: The small molecule nobiletin targets the molecular oscillator to enhance circadian rhythms and protect against metabolic syndrome. Cell Metab. 2016; 23: 610–621. PubMed Abstract | Publisher Full Text\n\nNohara K, et al.: Ammonia-lowering activities and carbamoyl phosphate synthetase 1 (Cps1) induction mechanism of a natural flavonoid. Nutr. Metab. (Lond.). 2015; 12: 23. PubMed Abstract | Publisher Full Text\n\nNohara K, et al.: Nobiletin fortifies mitochondrial respiration in skeletal muscle to promote healthy aging against metabolic challenge. Nat. Commun. 2019; 10: 3923. PubMed Abstract | Publisher Full Text\n\nNohara K, et al.: Cardiolipin Synthesis in Skeletal Muscle Is Rhythmic and Modifiable by Age and Diet. Oxidative Med. Cell. Longev. 2020; 2020: 5304768.\n\nKim E, et al.: Effects of the Clock Modulator Nobiletin on Circadian Rhythms and Pathophysiology in Female Mice of an Alzheimer’s Disease Model. Biomolecules. 2021; 11: 1004. PubMed Abstract | Publisher Full Text\n\nRakshit K, Matveyenko AV: Induction of Core Circadian Clock Transcription Factor Bmal1 Enhances beta-Cell Function and Protects Against Obesity-Induced Glucose Intolerance. Diabetes. 2021; 70: 143–154. PubMed Abstract | Publisher Full Text\n\nPetrenko V, et al.: In pancreatic islets from type 2 diabetes patients, the dampened circadian oscillators lead to reduced insulin and glucagon exocytosis. Proc. Natl. Acad. Sci. U. S. A. 2020; 117: 2484–2495. PubMed Abstract | Publisher Full Text\n\nNohara K, Nemkov T, D'Alessandro A, et al.: Coordinate Regulation of Cholesterol and Bile Acid Metabolism by the Clock Modifier Nobiletin in Metabolically Challenged Old Mice. Int. J. Mol. Sci. 2019; 20. PubMed Abstract | Publisher Full Text\n\nOyama Y, Bartman CM, Gile J, et al.: The Circadian PER2 Enhancer Nobiletin Reverses the Deleterious Effects of Midazolam in Myocardial Ischemia and Reperfusion Injury. Curr. Pharm. Des. 2018; 24: 3376–3383. PubMed Abstract | Publisher Full Text\n\nShinozaki A, et al.: Potent Effects of Flavonoid Nobiletin on Amplitude, Period, and Phase of the Circadian Clock Rhythm in PER2::LUCIFERASE Mouse Embryonic Fibroblasts. PLoS One. 2017; 12: e0170904. PubMed Abstract | Publisher Full Text\n\nWirianto M, et al.: The clock modulator Nobiletin mitigates astrogliosis-associated neuroinflammation and disease hallmarks in an Alzheimer's disease model. FASEB J. 2022; 36: e22186.\n\nYang X, et al.: Nobiletin Delays Aging and Enhances Stress Resistance of Caenorhabditis elegans. Int. J. Mol. Sci. 2020; 21. Publisher Full Text\n\nKim E, et al.: ROR activation by Nobiletin enhances antitumor efficacy via suppression of IκB/NF-κB signaling in triple-negative breast cancer. Cell Death Dis. 2022; 13: 374. PubMed Abstract | Publisher Full Text\n\nLellupitiyage Don SS, et al.: Nobiletin affects circadian rhythms and oncogenic characteristics in a cell-dependent manner. PLoS One. 2020; 15: e0236315. PubMed Abstract | Publisher Full Text\n\nChen Z, Yoo S-H, Takahashi JS: Development and therapeutic potential of small-molecule modulators of circadian systems. Annu. Rev. Pharmacol. Toxicol. 2018; 58: 231–252. PubMed Abstract | Publisher Full Text\n\nKoronowski KB, Sassone-Corsi P: Communicating clocks shape circadian homeostasis. Science. 2021; 371. PubMed Abstract | Publisher Full Text"
}
|
[
{
"id": "154583",
"date": "11 Nov 2022",
"name": "Shihoko Kojima",
"expertise": [
"Reviewer Expertise Circadian genomics",
"rhythmic gene expression",
"mouse",
"clock-controlled genes"
],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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:\nIn the review article titled “Circadian stabilization loop: the regulatory hub and therapeutic target promoting circadian resilience and physiological health”, the authors discuss various studies on the role of Rev-erbs and Rors on health and aging with the main focus being their function within the circadian clock. The authors give a well-balanced and comprehensive approach to discussing the importance of these genes in many different physiological pathways, such as immune response, metabolic disorders, cancer, and more. The authors provide extensive citations to support their discussion as well. We only have minor comments, and believe the article is suitable for approval.\nMinor Concerns:\nIn the second paragraph of the section titled “The circadian timing system and health implications”, Dbp and Nfil3 are not mentioned as part of the circadian clock, despite their known regulation of multiple clock genes via D-boxes.\n\nIn the third paragraph of the section titled “The stabilization loop”, the authors state “While Ror expressions display relatively moderate circadian oscillatory amplitude, Rev-erbs are among the most oscillatory genes (highest amplitude) in both protein and mRNA expression”. However, Rors themselves differ in amplitude with Rora expression showing little to no rhythmicity in most tissues. Meanwhile, Rorc expression is rhythmic and displays similar amplitude to Rev-erbs in some tissues/cell types1,2,3.\n\nThe nomenclature for mouse models should be superscripted (for example, Rorasg/sg rather than Rorasg/sg)\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": "9073",
"date": "12 Dec 2022",
"name": "Eunju Kim",
"role": "Author Response",
"response": "Point-by-Point Response We thank both expert reviewers for their thorough and thoughtful comments. Our response is as follows. Reviewer 1 In the review article titled “Circadian stabilization loop: the regulatory hub and therapeutic target promoting circadian resilience and physiological health”, the authors discuss various studies on the role of Rev-erbs and Rors on health and aging with the main focus being their function within the circadian clock. The authors give a well-balanced and comprehensive approach to discussing the importance of these genes in many different physiological pathways, such as immune response, metabolic disorders, cancer, and more. The authors provide extensive citations to support their discussion as well. We only have minor comments, and believe the article is suitable for approval. We thank the reviewer for the overall positive comments. Minor Concerns: 1. In the second paragraph of the section titled “The circadian timing system and health implications”, Dbp and Nfil3 are not mentioned as part of the circadian clock, despite their known regulation of multiple clock genes via D-boxes. Apologies for this omission. We have added Dbp and Nfil3 as additional core clock genes functioning in the oscillator on page 3. 2. In the third paragraph of the section titled “The stabilization loop”, the authors state “While Ror expressions display relatively moderate circadian oscillatory amplitude, Rev-erbs are among the most oscillatory genes (highest amplitude) in both protein and mRNA expression”. However, Rors themselves differ in amplitude with Rora expression showing little to no rhythmicity in most tissues. Meanwhile, Rorc expression is rhythmic and displays a similar amplitude to Rev-erbs in some tissues/cell types1,2,3. Thank you for this great comment. We have added a discussion text with these references to highlight the differences between the RORs (page 5, paragraph 2 in “the stabilization loop”). We thank the reviewer for this excellent suggestion. 3. The nomenclature for mouse models should be superscripted (for example, Rorasg/sg rather than Rorasg/sg) As suggested, we have superscripted the mouse model names. Thank you again."
}
]
},
{
"id": "154584",
"date": "11 Nov 2022",
"name": "Tsuyoshi Hirota",
"expertise": [
"Reviewer Expertise circadian biology",
"small molecules"
],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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, Kim, Yoo, and Chen beautifully and convincingly describe the relevance of REV-ERB and ROR nuclear hormone receptors as therapeutic targets of metabolic disorders, immune diseases, brain diseases, muscle pathologies, and cancer, as well as current challenges in the research, with special emphasis on natural compound nobiletin. This well-written review article covers the translation of the molecular clock mechanism for health and healthy aging, which is an important topic in the field. I strongly support the publication of this article, and a brief description of the following points would be useful for further understanding:\nAre the therapeutic effects of nobiletin dependent on both RORα and RORγ? As the authors described, isoforms of REV-ERB (α and β) and ROR (α, β, and γ) have different expression patterns and physiological functions. Therefore, it would be nice to discuss the possibility and potential of isoform-selective ligands as well.\n\nBecause nobiletin is a natural compound and other REV-ERB/ROR ligands are synthetic compounds, it would be nice to mention the merit (and demerit) of natural compounds compared to synthetic compounds.\n\nMinor points:\nPage 4, line 2: Drosophila to be italic. Page 5, “Therapeutic relevance of REV-ERBs and RORs” section, lines 2-3: “expression” is duplicated. Page 6, line 11: ZT may need an explanation. Page 6, the second paragraph, line 1: Rev-erba-/- to be italic. Please check whether references 60 (Emery and Clayton, 2001) and 137 (Nagoshi et al., 2004) are proper literature in the context.\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": "9074",
"date": "12 Dec 2022",
"name": "Eunju Kim",
"role": "Author Response",
"response": "Point-by-Point Response We thank both expert reviewers for their thorough and thoughtful comments. Our response is as follows. Reviewer 2 In this manuscript, Kim, Yoo, and Chen beautifully and convincingly describe the relevance of REV-ERB and ROR nuclear hormone receptors as therapeutic targets of metabolic disorders, immune diseases, brain diseases, muscle pathologies, and cancer, as well as current challenges in the research, with special emphasis on natural compound nobiletin. This well-written review article covers the translation of the molecular clock mechanism for health and healthy aging, which is an important topic in the field. I strongly support the publication of this article, and a brief description of the following points would be useful for further understanding: Thank you very much for the positive comments. Are the therapeutic effects of nobiletin dependent on both RORα and RORγ? As the authors described, isoforms of REV-ERB (α and β) and ROR (α, β, and γ) have different expression patterns and physiological functions. Therefore, it would be nice to discuss the possibility and potential of isoform-selective ligands as well. Thank you for this valuable comment. NOB shows robust binding to the LBDs of RORα and RORγ, with somewhat higher affinity for RORγ (PMID: 27076076). However, there is currently no functional evidence for a possible selectivity of NOB toward either ROR. We agree that potential isoform/subtype-selective ligands, such as those characterized for CRYs, would be valuable. We have added this discussion to the text on page 9. Because nobiletin is a natural compound and other REV-ERB/ROR ligands are synthetic compounds, it would be nice to mention the merit (and demerit) of natural compounds compared to synthetic compounds. Thank you for this excellent comment. NOB’s excellent safety profile is indeed a significant advantage over other synthetic ligands which may require extensive medicinal chemistry efforts before in vivo and clinical applications. Without making a direct comparison, we have added a sentence on page 10 to highlight this point. Thank you. Minor points: Page 4, line 2: Drosophila to be italic. As suggested, we have italicized the word. Page 5, “Therapeutic relevance of REV-ERBs and RORs” section, lines 2-3: “expression” is duplicated. As suggested, we corrected the sentence. Page 6, line 11: ZT may need an explanation. Apologies for this omission. We added the full name and explanation of ZT in the manuscript. Page 6, the second paragraph, line 1: Rev-erba-/- to be italic. As suggested, we have italicized the word. Please check whether references 60 (Emery and Clayton, 2001) and 137 (Nagoshi et al., 2004) are proper literature in the context. As suggested, we have removed these references as they are not immediately relevant as the reviewer pointed out. Thank you again."
}
]
}
] | 1
|
https://f1000research.com/articles/11-1236
|
https://f1000research.com/articles/11-1484/v1
|
12 Dec 22
|
{
"type": "Research Article",
"title": "Importance of social support for Indonesian stroke patients with depression",
"authors": [
"Nizar Yamanie",
"Aly Lamuri",
"Yuli Felistia",
"Oedojo Soedirham",
"Windhu Purnomo",
"Amal Chalik Sjaaf",
"Muhammad Miftahussurur",
"Nizar Yamanie",
"Aly Lamuri",
"Yuli Felistia",
"Oedojo Soedirham",
"Windhu Purnomo",
"Amal Chalik Sjaaf"
],
"abstract": "Background: Approximately one-third of stroke survivors experience depression at some point, which is linked to poor functional results and high mortality rate. Social support from family, friends, and the community is an intervening variable in stroke outcomes aside from the rehabilitation treatments that patients receive. This study assessed the importance of social support for stroke patients with depression and its relationship with patient rehabilitation. Methods: This quantitative study used a cross-sectional approach on stroke patients and their families based on data from the Social Security Administrator for Health (BPJS Kesehatan). One hundred and four participants were recruited using purposive sampling by including stroke patients who have used National Health Insurance (JKN) for stroke medications. Results: We found that instrumental, emotional, interactive, and information support contribute to lowering depressive symptoms. Instrumental support in the form of food availability, money, goods, and services had the highest coefficient value for reducing depression. Emotional support in the form of care and compassion had the second highest value in reducing depression. Further, interaction and informational support remain critical components of social support in reducing depression.\n\nConclusion: The support system plays a key role in decreasing the depression level in stroke survivors. The family and neighborhood have a significant impact on accelerating the rehabilitation process of stroke patients by providing support.",
"keywords": [
"Stroke",
"social interaction",
"informational support",
"non-communicable disease",
"depression."
],
"content": "Introduction\n\nStrokes greatly affect the lives of survivors and has been recognized as a main cause of disability.1 Long-term impairment of motor, sensory, and/or cognitive functions are experienced by stroke patients, and this is highly likely to correlate with social changes.2 Strokes have contributed to the death of 6.6 million people worldwide, including 3.3 million due to ischemic stroke, 2.9 million due to intracerebral hemorrhage, and 0.4 million due to subarachnoid hemorrhage.3,4 In Indonesia, approximately 10.9% of the population, over the age of 15, were affected by some form of stroke with the highest occurrence in East Kalimantan (14.7%) and Yogyakarta (14.6%).5,6\n\nPatients who have suffered a stroke and their caregivers are burdened with health, economic, and social consequences.7 Epidemiological studies have indicated that approximately 30% of stroke patients in the early or late stages develop post-stroke depression, which affects the rehabilitation motivation of patients, reduces the rehabilitation effect, and increases the burden of family care.8–10 Approximately one-third of stroke survivors experience depression at some point in their lives, which is linked to poor functional results and high mortality.11–13\n\nTo be able to live independently, patients must learn adaptation skills, including the ability to search for social resources. A stroke patient also need supports such as emotional, informational, instrumental, and social interaction support.14–16\n\nA patient who maintains meaningful social connections and actively engages in social activities successfully recovers and rejoins society. To interact or participate in social activities, the patients require physical abilities. To provide comprehensive healthcare, functional therapists should address the psychological requirements of stroke patients in addition to rehabilitation activities.17 This study aimed to assess the importance of social support among stroke patients with depression and the relationship between social support and patient rehabilitation.\n\n\nMethods\n\nA quantitative, cross-sectional approach was used in this study. The participants included stroke patients and their family members who were selected based on data from the Social Security Administrator for Health (BPJS Kesehatan). Using purposive sampling, we recruited 104 participants aged >30 years. The study period was from November 8th to December 15th 2021 and was conducted by Prof. Dr. Mahar Mardjono of the National Brain Center Hospital.\n\nEach of the participant complete the questionnaire. The data were distributed by the Social Security Administrator for Health.\n\nThe socio-demographic data that were assessed in this study were: age (30–39 years, 40–59 years, and >60 years), sex (male and female), highest education (primary, secondary, and higher education levels), last occupation (public sector, private sector, and retirement/unemployment), salary index (below 4.6 million and above 4.6 million rupiah – based on DKI Jakarta’s minimum standard salary), being a caregiver (yes or no), accompanied by family during check-ups (yes or no), the ownership of National Health Insurance (yes or no), transferred patients (yes or no), first medication (yes or no), stroke duration (>6 months, 7–12 months, and >1 year), returning to work after stroke (yes or no), the status of occupation post-stroke (full-time, part-time, and retirement/unemployment), social support (low, average, or high support),18 and work productivity (stagnant or decreased).\n\nTo measure the support system of caregivers, we used social support terminology, which is divided into emotional, informational, instrumental, and social interaction support (low, average, high). For depression levels, we used the Hamilton Rating Scale for Anxiety (HARS).\n\nThe data were analyzed using IBM SPSS Statistics version 26.0. (SPSS, Inc., Chicago, IL, USA; RRID:SCR_019096). For categorical variables, values were expressed as proportions and percentages, whereas for quantitative variables, means and standard deviations were reported. Fisher’s exact test was used for other variables. The statistical significance level was set at a p value of 0.05.\n\nIn this study, support systems were indirectly measured as reflective observable variables, including social, emotional, instrumental, informational, and social interaction support. A structural equation model (SEM) model was used to evaluate this relationship using lavaan package in the R project for statistical computing (R Foundation for Statistical Computing, Vienna, Austria; RRID:SCR_001905). Since all the observed variables were ordinal data (a categorized form of the measurement), we performed a polychoric correlation by applying a diagonally weighted least square (DWLS) estimator to the SEM model. Goodness of fit in the SEM model was measured using relative fit metrics, that is, the comparative fit index (CFI) and Tucker-Lewis index (TLI), and absolute fit metrics, namely the root mean square error of approximation (RMSEA) and standardized root mean residual (SRMR). For CFI and TLI, the thresholds for goodness of fit were >0.9 CFI, <0.08 for RMSEA, and <0.1 for SRMR. After confirming the latent construct of the support system, we further evaluated the relationship between the support system and depression scores by fitting a structural equation model (SEM).\n\n\nResults\n\nThe total number of participants in this study was 104 families each containing a stroke patient. The majority of participants were older than 60 years (50%), followed by those who were 40–59 years (46.2%). The study involved a higher number of male participants (67 patients, 64.4%) than female participants (35 patients, 35.6%). Most participants had secondary education (44.2%), followed by higher levels (41.3%). All working participants were employed in the private sector, and as many as 58 (55.8%) earned a monthly income over 4.6 million rupiah (67.3%).\n\nThe participants tended to have no caregivers during the stroke (93.3%). Approximately 89.4% of the participants had been visiting the medication center facility with their families. Nearly all participants were covered under National Health Insurance (98.1%), and 58.7% were classified as transferred from primary healthcare facilities. Approximately 89.4% of the participants were not on medication for the first time. In addition, 65.0% of participants decided not to work after experiencing a stroke. Further, 66.3% of the participants were retired or unemployed. Moreover, approximately 84.5% of the participants declared that their work productivity decreased after their stroke.\n\nThe socio-demographic characteristics of the participants are depicted in Table 1. First, with regard to family support, patients who received this support at a high level (89.4%) demonstrated a significant correlation with the decrease in depression (X2=52.9; p<0.01). Second, high emotional support (91.4%) also significantly influenced the decrease in depression (X2=27.2; p<0.01). Third, patients who received a high level of instrumental support from their families (83.4%) were more likely to have decreased depression levels (X2=56.7; p<0.01) that those who received low or an average level of support. Fourth, high-level informational support from the family (87.5%) tended to decrease depression levels among stroke patients (X2=9.3; p<0.01). Finally, high developmental support (89.4%) indicated a positive correlation (X2=104.0; p<0.01).\n\n\n\n- 30–39 years\n\n\n\n- 40–59 years\n\n\n\n- >60 years\n\n\n\n- Male\n\n\n\n- Female\n\n\n\n- Primary level\n\n\n\n- Secondary level\n\n\n\n- Higher level\n\n\n\n- Public sector\n\n\n\n- Private sector\n\n\n\n- Retirement/unemployment\n\n\n\n- <4.6 million\n\n\n\n- >4.6 million\n\n\n\n- Yes\n\n\n\n- No\n\n\n\n- Yes\n\n\n\n- No\n\n\n\n- Yes\n\n\n\n- No\n\n\n\n- Yes\n\n\n\n- No\n\n\n\n- First time\n\n\n\n- More than once\n\n\n\n- <6 months\n\n\n\n- 7–12 months\n\n\n\n- >1 year\n\n\n\n- Yes\n\n\n\n- No\n\n\n\n- Full-time\n\n\n\n- Part-time\n\n\n\n- Retirement/unemployment\n\n\n\n- Decrease\n\n\n\n- Stagnant\n\n\n\n- Low\n\n\n\n- Average\n\n\n\n- High\n\n\n\n- Low\n\n\n\n- Average\n\n\n\n- High\n\n\n\n- Very low\n\n\n\n- Low\n\n\n\n- Average\n\n\n\n- High\n\n\n\n- Low\n\n\n\n- Average\n\n\n\n- High\n\n\n\n- Low\n\n\n\n- Average\n\n\n\n- High\n\nInitially, we assessed a latent construct of the support system, reflected as the categorized variables of social support, emotional support, instrumental support, informational support, and recognition. The latent variable was assessed using a SEM model using a DWLS estimator, resulting in a fit model (CFI=1, TLI=1, RMSEA=0, and SRMR=0.05). The seemingly high CFI and TLI, along with a very low RMSEA, could indicate a small sample size and low correlation among the observed variables. Although the model is constrained to the sampled population, it provides the insight that all measures correspond to the latent construct of the support system. In the SEM model, social support was used as a reference for the other variables. All the variables positively contributed to the latent construct of the support system, indicating that all the instruments measured the same dimension. From the following table, we can conclude that instrumental support provides the highest contribution to the support system, whereas informational and social interaction support make the lowest contribution to the support system (Table 2).\n\n* Significant (p<0.05)\n\nSince the model returned a good fit, we determined how the latent construct of a support system corresponded to the measure of depression using a structural equation model (SEM) as depicted in Figure 1. Upon model fitting, the model indicated CFI=1, TLI=1, RMSEA=0, and SRMR=0.05, similar to the model fitness of the SEM model. The reflective behavior of each observed variable follows a trend similar to that of the SEM model, where instrumental support has the highest contribution and informational support has the lowest contribution. The estimated latent variable of social support is inversely proportional to depression, where each additional point in social support reduces the depression score by 0.2 points (p=0.042).\n\n\nDiscussion\n\nThis study found that socio-demographic factors, particularly age and sex, play a significant role in depression levels among stroke survivors. Most stroke patients are over 60 years of age. The findings of the present study are in line with the results of other research19–21 which demonstrated that the onset of a stroke mostly occurred among elderly individuals aged 60–80 years. In fact, age is a non-modified factor for ischemic stroke because of the changing anatomy of arteries, which tend to become narrower and harder with aging. Aside from age, we found that sex has also been classified as a predominant factor of strokes, in which males are more likely to be diagnosed with a stroke compared with females, similar to a previous study.19,20,22\n\nThis research found that a variety of support systems for stroke patients, including instrumental, emotional, information, and social interaction support, are inversely proportional to the level of depression. Instrumental support was the first significant support. Family instrumental support is a type of support directly provided by the family, such as loans for food, money, goods, and services.21 According to instrumental support is direct and tangible, such as lending money or alleviating the stress which accompanies performing tasks.23 This study found that instrumental support had a significant positive relationship with low levels of depression. This finding is in line with the findings of previous studies.24\n\nEmotional support has a significant relationship with low depression levels in stroke patients. Theoretically, people have a propensity to absorb the emotions and moods of others, which occurs through unconscious interactions.25 The close partners of patients, including caregivers and family members realize the anxiety and depression of the patients.26\n\nSocial interaction support is also positively correlated with depression levels in stroke patients, wherein the higher the social interaction support they received from the family, the lower their depression level. Supporting this finding, a study revealed that using social interaction support as a rehabilitation strategy to improve the quality of life of stroke patients is effective.27 Furthermore, stroke patients feel isolated, and their physical and psychological conditions will improve only if they obtain a high degree of social interaction support. Moreover, social interaction support is associated with a decline in depression symptoms,28 and prevents stroke recurrence.29,30\n\nInformational support was highly likely to be associated with low depression levels in stroke patients. Information support is defined as the provision of knowledge to seek help in solving practical problems. If this form of support is combined with emotional and instrumental support, it is referred to as the quality of social support. concluded that informational support aimed to fulfil the psychological and physiological needs of stroke patients and can be gained through social interaction support.8 In contrast, one study indicated that the majority of stroke patients did not receive appropriate informational support, especially from their families.31\n\nFurthermore, several important limitations of this study must be considered. Firstly, this was a cross-sectional study, which did not imply causality. Secondly, a recall memory bias could have occurred when the health history of patients was enquired. However, the data were representative of the data obtained from the National Brain Center Hospital.\n\n\nConclusions\n\nInstrumental, emotional, interactive, and information support play key roles in decreasing depression in stroke patients. Families of patients have a significant impact on providing these types of support. Hence, rehabilitation of stroke patients involving the family is important to increase the possibility of preventing depression due to a stroke. Social interaction should be the predominant support for stroke patients since this support can not only improve physical rehabilitation but also the psychological conditions of patients. The findings of this study suggest that practitioners should educate the families of stroke patients on how to strengthen the mental health of the patients using a combination of informational and social interaction support. We suggest a public policy to initiate the establishment of a social support center for stroke patients for social therapy and rehabilitation to improve the condition and quality of life.\n\n\nConsent\n\nThe authors certify that the participants provided informed consent after the objectives of the study were explained. Before recruiting participants, we provided detailed information and confirmed their agreement to participate by obtaining their signatures in the informed consent form. The families have been informed that names and initials will not be published, and due efforts will be made to conceal the identities of patients, but anonymity cannot be guaranteed.\n\n\nEthical approval\n\nThis study obtained ethical approval from Prof. Dr. Mahar Mardjono of the National Brain Centre Hospital Research Ethics Committee (reference number LB.02.01/KEP/089/2021).\n\n\nAuthor Contributions\n\nNizar Yamanie: Conceptualization, Methodology, Writing—original draft preparation, Writing—review and editing; Aly Lamuri: Conceptualization, Writing—original draft preparation, Writing—review and editing; Yuli Felistia: Conceptualization, Methodology, Resource, Writing—review and editing; Oedojo Soedirham: Conceptualization, Methodology, Data curation, Validation, Writing—original draft preparation; Windhu Purnomo: Conceptualization, Methodology, Data accuracy, Writing—original draft preparation; Amal Chalik Sjaaf: Conceptualization, Methodology, Writing—original draft preparation; Muhammad Miftahussurur: Conceptualization, Supervision, Writing—original draft preparation.",
"appendix": "Data availability\n\nAll data underlying the results are accessible through Mendeley data storage.\n\nData for: Importance of social support for Indonesian stroke patients with depression. https://doi.org/10.17632/xpzcnf3998\n\nThe project contains the following underlying data:\n\n- data (The directory containing all input data):\n\n○ clean.csv (De-identified primary data)\n\n○ sub-data.csv (Subset of data used for fitting a SEM)\n\n- RData (The directory containing R session objects):\n\n○ 01-linear-model. RData (Objects from script 01-linear-model. R)\n\n○ 02-pca. RData (Objects from script 02-pca. R)\n\n○ mod-cfa-support-system. Rds (The CFA model)\n\n○ mod-linreg. Rds (The linear regression model)\n\n○ mod-pca-support-depression. Rds (The PCA model)\n\n○ mod-sem-support-depression. Rds (The SEM model)\n\nData repository name: https://doi.org/10.17632/xpzcnf3998\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).\n\n\nReferences\n\nWHO: Global Status Report on Noncommunicable Diseases 2014. World Health Organization;2014.Reference Source\n\nOECD: Health at a Glance 2007: OECD Indicators. Joint OECD/Korea Regional Centre on Health and Social Policy;2008. Publisher Full Text\n\nAHA: Heart Disease and Stroke Statistics Update Fact Sheet Global Burden of Disease. American Heart Association;2021.\n\nIHME: Global Burden of Disease (GBD). Institute for Health Metrics and Evaluation;2019.\n\nKemenkes RI: Infodatin: Stroke Don’t Be The One. Kementerian Kesehatan Republik Indonesia. 2019.\n\nKemenkes RI: Laporan Hasil Riset Kesehatan Dasar (Riskesdas) Indonesia tahun 2018. Kementerian Kesehatan Republik Indonesia. 2018; 182–183.\n\nKitoko GMB, Vivalya BMN, Vagheni MM, et al.: Psychological burden in stroke survivors and caregivers dyads at the rehabilitation center of Kinshasa (Democratic Republic of Congo): A cross-sectional study. J Stroke Cerebrovasc Dis. 2022; 31(6): 106447. PubMed Abstract | Publisher Full Text\n\nNorthcott S, Moss B, Harrison K, et al.: A systematic review of the impact of stroke on social support and social networks: associated factors and patterns of change. Clin Rehabil. 2016; 30(8): 811–831. PubMed Abstract | Publisher Full Text\n\nLin FH, Yih DN, Shih FM, et al.: Effect of social support and health education on depression scale scores of chronic stroke patients. Medicine (Baltimore). 2019; 98(44): e17667. PubMed Abstract | Publisher Full Text | Free Full Text\n\nFadhilah H, Permanasari VY: Beban ekonomi yang ditanggung pasien dan keluarga akibat penyakit stroke: studi literatur. Ber Kedokt Masy. 2019; 35(6): 193–197.\n\nKutlubaev MA, Hackett ML: Part II: predictors of depression after stroke and impact of depression on stroke outcome: an updated systematic review of observational studies. Int J Stroke. 2014; 9(8): 1026–1036. PubMed Abstract | Publisher Full Text\n\nBartoli F, Lillia N, Lax A, et al.: Depression after stroke and risk of mortality: a systematic review and meta-analysis. Stroke Res Treat. 2013; 2013.\n\nHackett ML, Pickles K: Part I: frequency of depression after stroke: an updated systematic review and meta-analysis of observational studies. Int J Stroke. 2014; 9(8): 1017–1025. PubMed Abstract | Publisher Full Text\n\nBaumann M, Lurbe-Puerto K, Bucki B: Harmony and divergences in couples. Family and social implications two years post-stroke. Sc. Ann Alexandru Ioan Cuza Univ New Ser Sociol Soc Work Sect. 2012; 5: 155–173.\n\nBlessing M, Oluwagbemiga O: Effectiveness of social support in coping with stroke by medically ill patient in Ibadan. Int J Neurorehabilitation. 2017; 04(04): 281–2376. Publisher Full Text\n\nGlass TA, Matchar DB, Belyea M, et al.: Impact of social support on outcome in first stroke. Stroke. 1993; 24(1): 64–70. Publisher Full Text\n\nBaumann M, Couffignal S, Le Bihan E, et al.: Life satisfaction two-years after stroke onset: the effects of gender, sex occupational status, memory function and quality of life among stroke patients (Newsqol) and their family caregivers (Whoqol-bref) in Luxembourg. BMC Neurol. 2012; 12(1): 1–11. Publisher Full Text\n\nCohen S, Wills TA: Stress, social support, and the buffering hypothesis. Psychol Bull. 1985; 98(2): 310–357. Publisher Full Text\n\nKissela BM, Khoury JC, Alwell K, et al.: Age at stroke: temporal trends in stroke incidence in a large, biracial population. Neurology. 2012; 79(17): 1781–1787. PubMed Abstract | Publisher Full Text | Free Full Text\n\nYang Y, Yang Y, Jin G, et al.: The prevalence of stroke and related risk factors among residents aged≥ 40 years in Chongqing, Southwest China. J Public Health (Bangkok). 2021; 29(6): 1423–1432. Publisher Full Text\n\nKurniawan MB, Wibowo TA: Hubungan antara Dukungan Informasi Keluarga dengan Depresi pada Pasien Pasca Stroke di PUSKESMAS Remaja Samarinda. Borneo Student Res. 2020; 1(2): 1280–1286.\n\nJun HJ, Kim KJ, Chun IA, et al.: The relationship between stroke patients’ socio-economic conditions and their quality of life: the 2010 Korean community health survey. J Phys Ther Sci. 2015; 27(3): 781–784. PubMed Abstract | Publisher Full Text | Free Full Text\n\nSarafino EP, Smith TW: Health Psychology: Biopsychosocial Interactions. John Wiley & Sons;2014.\n\nDewi CM, Darliana D: Dukungan keluarga dengan depresi pada pasien pasca stroke. Idea Nurs J. 2017; 8(3).\n\nHerrando C, Constantinides E: Emotional contagion: a brief overview and future directions. Front Psychol. 2021; 12: 712606. PubMed Abstract | Publisher Full Text | Free Full Text\n\nGoodman CR, Shippy RA: Is it contagious? Affect similarity among spouses. Aging Ment Health. 2002; 6(3): 266–274. PubMed Abstract | Publisher Full Text\n\nVenna VR, Xu Y, Doran SJ, et al.: Social interaction plays a critical role in neurogenesis and recovery after stroke. Transl Psychiatry. 2014; 4: e351. PubMed Abstract | Publisher Full Text | Free Full Text\n\nPark EY, Kim JH: An analysis of depressive symptoms in stroke survivors: Verification of a moderating effect of demographic characteristics. BMC Psychiatry. 2017; 17(1): 132. PubMed Abstract | Publisher Full Text | Free Full Text\n\nElloker T, Rhoda AJ: The relationship between social support and participation in stroke: A systematic review. African J Disabil. 2018; 7: 7. PubMed Abstract | Publisher Full Text | Free Full Text\n\nLiu FY, Chen CH:Using social interaction in rehabilitation to improve stroke patients motivation. International Conference on Applied Human Factors and Ergonomics. Springer; 2018; 109–120.\n\nSaprianto S, Amalia LR, Aini F, Sukarno S: Relationship Of Family Support To Post Stroke Depression Levels In Outpatient Installation Dr. Kariadi Semarang. Proceeding International Conference On Health, Social Sciences And Technology. 2021; 1: 115–117."
}
|
[
{
"id": "242303",
"date": "21 Mar 2024",
"name": "Mahnaz Khatiban",
"expertise": [
"Reviewer Expertise \"Qualitative and Quantitative Research in Health-Related Subjects.\"InsertRetryMake it persuasive"
],
"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\nDear Editorial Board, Thank you for sending the manuscript entitled \"Importance of social support for Indonesian Stroke Patients with depression\" for review. This manuscript reports the correlation between social support for stroke patients with depression and patient rehabilitation among stroke patients and their families. In general, I liked the topic of the study, as social support is a crucial variable in stroke outcomes. However, I believe there are some areas where the author could provide further clarification. Please consider addressing the following details:\nTitle:\nPlease modify the title to “Correlation between social support and depression for Indonesian stroke patients: A cross-sectional study” in the title.\nAbstract:\nPlease provide the exact aims of the study. What is your meaning of importance in the “This study assessed the importance of social support for stroke patients with depression and its relationship with patient rehabilitation”? I suggest writing: for example: the correlation between social support for stroke patients with their depression and rehabilitation. Please provide some information about the questionnaires you applied. Please address exactly who was your study population. were they the patients, their families, or both? How did you calculate your sample size? Although you have mentioned that you used purposive sampling, I cannot find the purpose of sampling. So, add some reasonable purposes, for example: the patient’s depression was confirmed by their physician or something like that. The result part: You have conducted a correlational or cross-sectional study, which indicates that you should use appropriate connecting words while writing the results. Rather than stating the sentence \"Emotional support in the form of care and compassion had the second highest value in reducing depression\", it is recommended to frame it as people with stroke who reported less depression also reported receiving more emotional support. This will help in presenting your results more effectively. Please rewrite the conclusion.\nKeywords:\nPlease provide the most relevant and specialized keywords according to MeSH.\nIntroduction\nIt would be beneficial to provide a clear reasoning for examining the correlation between social support and depression in stroke patients. This involves explaining why this correlation is important and relevant, especially in the Indonesian context. A brief discussion on the potential impact of social support on stroke recovery and patient well-being would help strengthen the justification for the study. At the end of the introduction, the authors’ assumptions are not clearly explained. The readers wouldn’t figure out why they selected these concepts for their study. In my opinion, the introduction could focus more clearly on the rationale for this study.\nMethod\nPlease explain your settings in your country. The authors have mentioned: “The participants included stroke patients and their family members who were selected based on data…”. But, I cannot distinguish the results of patients from family members. Who were the study participants? Patients? Family members? Or both?\nSampling\n\nWhat was the reason that you chose only >30 years old participants? Although you mentioned using purposive sampling, the purpose was not clear. Please clarify the purpose, for instance, the physician's confirmation of the patient's stroke, duration of disease, and having no comorbidity diseases such as diabetes, or CHF, …. Why didn't use a more reliable sampling, for example, cluster or categorical sampling? Is there only one hospital in Indonesia for stroke patients? If there are several hospitals, explain the reason for choosing one of them, National Brain Center Hospital. Why did you choose purposive sampling for selecting the participants? It seems you had the sampling framework that makes it easy to use probability sampling.\nSample size\nHow did you calculate your sample size? Did all of the participants respond to the questionnaires? Was there any reduction, incomplete or nonresponding questionnaires?\n\nMeasures\nThe Hamilton Rating Scale for Anxiety (HRSA) is a questionnaire specifically designed to measure anxiety levels and is not recommended for assessing depression. Do you happen to know of any sources that suggest using this scale for measuring depression?\n\nCan you please provide the readers with information about the questionnaire you use to assess social support and its dimensions? In which language were your questionnaires? How did you ensure their validity and reliability? How did you apply the questionnaire? By mail?\nData collection\nHow did you collect the data? By mail, phone, or in presence? Where did you reach the patients?\nData analysis\nAccording to the authors, the analysis is based on social support, which involves emotional, informational, instrumental, and social interaction support, categorized as low, average, and high. As a standard questionnaire was not employed, the analysis cannot be considered valid.\nResults\nIn the results section of the study, the author stated that there were a total of 104 families, each with a stroke patient. However, the study had a higher number of male participants (67 patients, 64.4%) than female participants (35 patients, 35.6%). This has left me confused about who the participants in the study were. Again, as the outcomes of depression are based on the anxiety scale and social support was not accomplished by a standard questionnaire, the results are not valid. Where are the results of rehabilitation? And its scale?\nDiscussion\nThe findings related to depression cannot be deemed valid because they were based solely on the anxiety scale, and social support was not evaluated using a standardized questionnaire. Therefore, the discussion and conclusion are questionable.\n\nIs the work clearly and accurately presented and does it cite the current literature? No\n\nIs the study design appropriate and is the work technically sound? No\n\nAre sufficient details of methods and analysis provided to allow replication by others? No\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nNo\n\nAre all the source data underlying the results available to ensure full reproducibility? Partly\n\nAre the conclusions drawn adequately supported by the results? No",
"responses": []
}
] | 1
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https://f1000research.com/articles/11-1484
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https://f1000research.com/articles/11-1062/v1
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16 Sep 22
|
{
"type": "Systematic Review",
"title": "Prevalence of musculoskeletal disorders among dental healthcare providers: A systematic review and meta-analysis",
"authors": [
"Deepika Chenna",
"Kalyana C Pentapati",
"Mathangi Kumar",
"Medhini Madi",
"Hanan Siddiq",
"Deepika Chenna",
"Mathangi Kumar",
"Medhini Madi",
"Hanan Siddiq"
],
"abstract": "Background: Work-related musculoskeletal disorders (MSD) are common in dentistry due to the prolonged static work involved during patient care, making dental health care personnel vulnerable to musculoskeletal complaints. We aimed to pool the prevalence estimates of MSD among various dental healthcare providers, including dentists, dental students, dental hygienists, and auxiliaries. Methods: A systematic search of five databases was performed (Scopus, Embase, CINAHL, Web of Science, Dentistry & Oral Sciences Source). The studies that reported the prevalence of MSD among dental healthcare workers and those written in English were selected. Screening and data extraction were performed by two review authors independently. Discrepencies were resolved by another review author. Risk of bias assessment was done using a nine-item questionnaire developed by Hoy et al. Pooled estimates were calculated using meta-analysis of proportions (random effects model). Results: Among the 3090 publications screened, 234 publications were included for full-text screening. Meta-analysis was performed for 89 estimates from 88 publications. Females showed significantly higher prevalence [OR = 1.42 (95% CI = 1.09–1.84); I2 = 66.02; N = 32]. The analysis yielded a pooled estimate of 78.4% (95% CI = 74.8–82). The meta-regression showed similar prevalence over the years (Coefficient: 0.001; P-value: 0.762). Conclusions: A high prevalence of MSD was noted among dental healthcare providers, with about seven out of ten having experienced MSD in the past. This emphasizes the need for awareness and adoption of appropriate ergonomic postures by dental healthcare providers from early in their careers to minimize work-related MSD.",
"keywords": [
"musculoskeletal disorders",
"workplace",
"dentist",
"dental students",
"dental auxiliary",
"systematic review"
],
"content": "Introduction\n\n“Musculoskeletal disorders (MSD) are injuries to the human support system of muscles, ligaments, tendons, nerves, blood vessels, bones, and joints” (https://www.cdc.gov/). Such injuries resulting due to occupation or work-related exposure are termed work-related MSD. Work-related MSD is common in dentistry due to the prolonged static work involved during patient care, making dental health care personnel vulnerable to musculoskeletal complaints. Moreover, the current lifestyle practices make the onset of such problems likely at an early stage of life. MSD includes pain, discomfort, or limitation in a range of activities in the head, neck, shoulders, arms, wrists, fingers, elbows, upper and lower back, buttocks, thighs, feet, ankle, etc.\n\nMSD among dental healthcare personnel can potentially impact the individual and the community. Literature has shown a decrease in work efficiency, stress, poor sleep quality, multisite pain, frequent absenteeism, and/or early retirement resulting in loss of workforce.1,2 The preventive strategies adopted to mitigate MSD are massage treatments, increased physical activity, adopting ergonomically designed equipment, maintaining correct postures, and using complementary and alternative medicine.3,4\n\nThe studies on self-reported MSD have reported a high prevalence among dental healthcare personnel.5–10 Studies have also evaluated the associated risk factors of MSD among dentists,7,11–19 dental hygienists,6,20,21 and dental students.22,23 Increasing age, gender (female), comorbidities, prolonged working hours, increased patient load, lack of physical exercise, non-usage of loupes, stress, lack of breaks between patients, awkward postures, administrative work, vibration, and repetition were some of the reported risk factors of MSD.4,24 A few literature reviews and meta-analysis on these conditions have reported a high prevalence among dental healthcare personnel.25–31 However, there was no attempt to study the overall prevalence estimates of MSD burden among various dental healthcare providers, including dentists, dental students, dental assistants, and auxiliaries at a global level. Hence, we aimed to pool the estimates of the MSD burden among dental healthcare providers.\n\n\nMethods\n\nThe studies that reported the overall prevalence of MSD among dental healthcare personnel (dentists, dental students, hygienists, or dental auxiliaries), and the studies written in English were included. The studies reported as commentaries, letters, or conference abstracts were excluded. The protocol was registered with INPLASY (DOI: 10.37766/inplasy2021.5.0100).32\n\nA systematic search in five databases (Scopus (RRID:SCR_022706), Embase (RRID:SCR_001650), CINAHL (RRID:SCR_022707), Web of Science (RRID:SCR_022706), Dentistry & Oral Sciences Source (RRID:SCR_022705)) from inception to 5 August 2021 was performed. The keywords used were “dentist OR dental hygienist OR dental personnel OR dental student” AND “musculoskeletal disease OR musculoskeletal disorder OR occupational disease OR work-related musculoskeletal disorder.” Suitable filters (reports on humans, research articles) for each database were applied.\n\nThe search was imported to Rayyan, a web-based application (RRID:SCR_017584).33 The screening and data extraction were done by two review authors independently (MK and MM). Disagreements were arbitrated by another review author (PKC).\n\nRisk of bias (RoB) assessment\n\nAll studies were assessed using the 10 item Quality Assessment Checklist for Prevalence Studies questionnaire34 by two review authors independently (HS and PKC). Disagreements were arbitrated by another review author (CD). Each question has two levels, low risk (0) and high risk (1). The total of all nine questions was used to categorize the studies as “low (0–3), moderate (4–6), or high risk (7–9)”.\n\nThe variables for data extraction included study details such as authors, year, country, continent, study design, sample size, type of participants (dentist or dental students, or dental auxiliaries), age distribution, sex distribution, the overall prevalence of MSD at maximum recall along with lifetime, annual, one-week prevalence, gender and site-specific estimates.\n\nDue to variation in the reporting of the prevalence of MSD among the included studies, the prevalence estimates at the maximal follow-up were used to calculate the pooled estimates of MSD. Measures of heterogeneity (Q and I2) were calculated. A random-effects model (restricted maximum likelihood estimation method) was used to calculate the prevalence estimates using the OpenMeta[Analyst] software for Windows 8 (Metafor Package 1.4, 1999) (RRID: SCR_022698). Time trends of MSD were evaluated using meta-regression. A sub-group analysis based on the continent, country, type of dental personnel, site of MSD, and sex was performed. A funnel plot was used to evaluate the publication bias. Complete data for the analysis can be accessed at Mendeley datasets.35\n\n\nResults\n\nA comprehensive systematic search of five databases (Scopus (1080), Embase (592), CINAHL (728), Web of Science (514), Dentistry & Oral Sciences Source (750)) yielded a total of 3664 articles. Reviews, conference proceedings, case reports, clinical trials, studies on ergonomics, quality of life, burnout, etc. letters, magazine reports, work related hazards other than MSD, studies among health professionals other than dentists were excluded (n = 2856). A further 146 publications were excluded after screening the full-text. Meta-analysis was performed for 89 estimates (Table 1 and Figure 1).\n\nThe prevalence of MSD ranged from 19.4 to 100%. Only seven publications showed less than than 50% of MSD.17,36–41 More than one-quarter (n = 24) of the included publications reported more than 90% prevalence.5–12,16,18,23,42–54 One fourth of the studies (n = 21) reported a lifetime prevalence,3,37,39,44,45,49,51,53–66 while only eight studies reported a one-week prevalence.8,18,19,22,42,53,54,67 Most of the included studies reported a one-year prevalence (n = 65) (Table 1).\n\nMost of the studies reported the age distribution of the participants (n = 61), while 14 studies reported only the age range of the participants. Prevalence estimates could not be calculated as there was substantial variation in age grouping.\n\nMost of the studies reported the gender distribution of the participants (n = 80). Only one-third of the studies (n = 32) reported gender-specific estimates. The pooled prevalence of MSD among males and females was 72.4% (95% CI = 65.2–79.6) and 77.4% (95% CI = 69.4–85.4) respectively6,7,10,12,13,16,18,22,23,38,39,41,53,56,58,59,62,67–80 (Table 2). Females had significantly higher estimates of MSD than males (OR = 1.42) (Figure 2).\n\nOnly a few studies were reported from North America (n = 7),37,43,49,52,63,81,82 South America (n = 4),19,50,77,83 and Australia (n = 4),14,68,84,85 while only one study was reported from Africa (n = 1).86 Most of the studies were from Asia3,5–10,12,15–18,22,23,36,38,40,41,45,46,48,51,55–62,64–66,69–71,73–76,78,79,87–100 and Europe11,13,39,42,44,47,53,54,67,72,80,101–105 (Table 2). Countries with more than three studies were included for the sub-group analysis. The highest pooled prevalence was seen in Malaysia, and the lowest pooled prevalence was seen in Greece.\n\nOut of the 88 studies included, only four studies had a moderate RoB.22,52,57,60 The pooled estimates for studies with low and moderate RoB were 79% and 74% (Table 2).\n\nThe commonly reported sites were the neck, back, lower back, shoulder, upper back, and wrists. The least affected sites were thighs, legs, arms, feet, and ankles (Table 3).\n\nThere was high heterogeneity among the included studies, as evidenced by Q and I2 statistics. The model yielded a pooled estimate of 78.4% (Figure 3), and sensitivity analysis did not show any change in the overall estimate. The meta-regression showed no change in the trend of MSD (Coefficient: 0.001; 95% CI: -0.004 to 0.006) (Figure 4). Asymmetry was noted in the funnel plot (p < 0.001) (Figure 5).\n\n\nDiscussion\n\nMSD’s result in pain, discomfort, or limitation in the range of movement. They are preventable conditions often due to poor ergonomic postures adopted by dental health care providers. We aimed to pool the estimates of MSD among dental healthcare providers. Eighty-eight publications recorded a comprehensive assessment of all body areas and reported the overall prevalence of MSD. The estimates needed to be evaluated carefully due to the high heterogeneity. The overall estimate was 78%, which was much higher than nationwide surveys.37,80 However, extensive surveys of dentists from India and Lithuania have reported similar or higher prevalence estimates.3,9,101 Therefore, it is clear that dental professionals have quite a higher prevalence of MSD. Age-specific prevalence estimates could not be estimated due to a lack of standardized age groups or specific prevalence estimates. It was found that females showed higher prevalence estimates than males. Although the number of studies that reported gender distribution was high, only one-third of these studies reported gender-specific estimates of MSD.\n\nThe prevalence estimates were similar across the continents. The highest number of studies were reported from the Asian continent. The highest number of studies were from India,3,5,9,10,18,38,51,55,56,59,60,64,65,69,74,75,78,88,97,99 followed by the US,37,43,49,52,81,82 Iran,15,16,48,66,71,89 and Turkey.36,40,45,61,70,87 Studies from Malaysia7,95,98 reported the highest prevalence estimates among various countries, followed by Iran,15,16,48,66,71,89 Sweden,42,102 Australia,14,84,85 Brazil,19,50,77,83 and the US.37,43,49,52,81,82 There was not much variation in the prevalence estimates among the dentists, dental auxiliaries, and dental students. These observations suggest that all types of dental healthcare providers globally suffer from MSDs due to prolonged static postures. Over three decades, there was no significant change in the trend of MSD, indicating a consistently higher prevalence, highlighting the need to incorporate ergonomics into the dental curriculum.\n\nThere was substantial inconsistency in the assessment of prevalence estimates among the studies. The Nordic/standardized Nordic questionnaire was the most commonly used tool to assess MSDs. A few studies used generic questionnaires and single-item questions without adequate validity and reliability. Moreover, the studies used various time recall periods (lifetime, one year, six months, one month, and one week) to assess the prevalence estimates. The studies that used lifetime or extended recall periods might have included pre-existing MSDs that may not be work-related, which could have diluted the estimates of MSD.\n\nMSD can arise from various reasons, and there was a lack of clarity in most of the studies. Only one study explicitly recorded the estimates before and after joining the dental profession.58 There was a general lack of clarity on the estimates reported for various body parts (shoulders, hands, elbow, wrists, legs, ankles, hips, fingers, toes). The studies reported right, left, and bilateral prevalence estimates of MSD without detailing the prevalence for each site. MSD in such areas could have been reported as unilateral and bilateral rather than right, left, and bilateral estimates. Furthermore, there was no uniformity in the evaluation of site-specific assessments among the studies included (e.g. lack of clarity on the terms hand and arms).\n\nThe strength of this review is the inclusion of studies that reported the overall estimates of MSD, including many databases, all types of dental healthcare personnel, overall, lifetime and annual estimates, sub-group analysis, gender, and site-specific prevalence estimates. A few limitations were observed in our study. They are the exclusion of studies published in other languages, lack of age-specific prevalence estimates, lack of differentiation between work-related and pre-existing MSDs, causes of MSDs due to inadequate reporting in primary studies, use of self-reported measures of MSD rather than objective measures, and exclusion of studies with no comprehensive assessment or overall estimates of MSD.\n\nThe additional confounding factors related to lifestyle (sedentary lifestyle, lack of regular physical exercise, and other extra-curricular activities) could significantly influence the onset and duration of MSD. Furthermore, the number of clinical working days/week, working hours/day, type and duration of procedures, specialization, number of patients/days, remedial measures, and history of MSD in the past could also substantially impact the estimates of MSD. These inconsistencies in the included studies could have influenced the overall prevalence of MSD.\n\n\nConclusions\n\nMSD among dental healthcare personnel is widespread and mostly chronic. Seven out of ten dental healthcare providers could have experienced MSD in the past. However, the severity and self-limiting nature of MSD cannot be underestimated. Awareness, adoption, and maintenance of appropriate ergonomic postures should be encouraged at dental schools and early in the career. Future studies should use the “Strengthening the Reporting of Observational Studies in Epidemiology (STROBE)” guidelines and use validated questionnaires for reporting MSD.\n\n\nData availability\n\nMendeley Data: Underlying data for ‘Musculoskeletal disorders among dental health care professionals’. https://www.doi.org/10.17632/2ttwfmzm9n.235\n\nMendeley Data: PRISMA checklist for ‘Musculoskeletal disorders among dental health care professionals’. https://www.doi.org/10.17632/2ttwfmzm9n.235\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0)",
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PubMed Abstract | Publisher Full Text\n\nKumar M, Pai KM, Vineetha R: Occupation-related musculoskeletal disorders among dental professionals. Med. Pharm. Reports. 2020; 93(4): 405–409. PubMed Abstract | Publisher Full Text\n\nAhmad NS, Abdullah AAA, Thyng OK, et al.: Musculoskeletal Disorders Among Dental Students. J. Res. Med. Dent. Sci. 2020; 8(3): 32–38.\n\nBhuvaneshwari S, Shveta J, Kaur J, et al.: Assessment of Various Dental Occupational Hazards and Safety Measures among Dentists of Odisha, India. J. Contemp. Dent. Pract. 2021; 21(10): 1165–1169. PubMed Abstract | Publisher Full Text\n\nSenosy SA, Anwar MM, Elareed HR: Profession-related musculoskeletal disorders among Egyptian physicians and dentists. J. Public Heal. 2020; 28(1): 17–22. Publisher Full Text\n\nPuriene A, Aleksejuniene J, Petrauskiene J, et al.: Self-reported occupational health issues among Lithuanian dentists. Ind. Health. 2008; 46(4): 369–374. PubMed Abstract | Publisher Full Text\n\nRundcrantz BL, Johnsson B, Moritz U: Pain and discomfort in the musculoskeletal system among dentists. A prospective study. Swed. Dent. J. 1991; 15(5): 219–228. PubMed Abstract\n\nKerosuo E, Kerosuo H, Kanerva L: Self-reported health complaints among general dental practitioners, orthodontists, and office employees. Acta Odontol. Scand. 2000; 58(5): 207–212. PubMed Abstract | Publisher Full Text\n\nZoidaki A, Riza E, Kastania A, et al.: Musculoskeletal disorders among dentists in the Greater Athens area, Greece: risk factors and correlations. J. Public Health (Bangkok). 2013; 21(21): 163–173. Publisher Full Text\n\nŠćepanović D, Klavs T, Verdenik I, et al.: The Prevalence of Musculoskeletal Pain of Dental Workers Employed in Slovenia. Work Heal. Saf. 2019; 67(9): 461–469. PubMed Abstract | Publisher Full Text"
}
|
[
{
"id": "151604",
"date": "13 Oct 2022",
"name": "Athira Nandakumar",
"expertise": [
"Reviewer Expertise Epidemiologist"
],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nIntroduction: The authors showed the high prevalence of Musculoskeletal disorder (MSD) among dental health care providers in a worldwide scenario. MSD-related problems seem to be a significant issue for this profession and this is an already known problem. The authors tried to put up possibly all available publications related to MSD and did this attempt. However, certain changes in the manuscript and corrections to spelling mistakes or typos are recommended.\nThe exact definition of MSD is missing.\nMethods: 1) Inclusion Criteria: Question: Is there any previously published systematic review included in this study? Question: Hygienists mentioned here are missing in Table 2. Are they included in auxiliaries? Question: What kind of studies were included - Cross-sectional/cohort/case-control? Question: Were there any self-reported studies?\n2) RoB: Question: What is the cutoff point used in this study? It is not mentioned that some cut-off point was used here. Question: How many articles were excluded after RoB calculations?\n3) Results: Question: Meta-analysis was performed for 89? Or 88? Question: What is the reason for the exclusion of 146 publications after the full screening of the full text?\n4) Prevalence: There is a repeated word “than than”.\n5) Geographic distribution: Question: The authors mentioned that Australia (4), Africa (1), and north and south America (7,4) why not Asia and Europe (N=?)? It may be better to revise this “One study from Africa (N=1)\".\n6) Figure 1: Question: Where is the N=89 in this chart? Question: What are the other sources used to identify additional records?\n7) Table 2: Question: Why is Africa not mentioned here? Question: Malaysia has the highest estimates. What is the reason? Is it due to the inappropriate definition of MSD in Malaysia? Question: The estimate for one year (0.82) is more than for a lifetime (0.78); What may be the possible reason? Question: What is the definition of “mixed”? Question: Dental auxiliaries seem to have more difficulties with MSD with an estimate of 0.83 (0.69-0.97). Is it not because 2-3 categories are grouped together?\n8) Limitation: Question: It’s mentioned that “the exclusion of studies published in other languages”; does it mean other than English?\nDiscussion: Question: What was the national survey estimate? Mentioned in references 37,80? Better to mention Greek and Czech surveys. Question: Reference 37, is it a national survey? Question: What was the prevalence of the studies that used the Nordic questionnaire?\n\nAre the rationale for, and objectives of, the Systematic Review clearly stated? Yes\n\nAre sufficient details of the methods and analysis provided to allow replication by others? Partly\n\nIs the statistical analysis and its interpretation appropriate? Yes\n\nAre the conclusions drawn adequately supported by the results presented in the review? Yes",
"responses": [
{
"c_id": "8917",
"date": "21 Oct 2022",
"name": "Kalyana Pentapati",
"role": "Author Response",
"response": "We thank the reviewer for their effort in reviewing this manuscript. Please find responses for the queries. The changes that are required to be done in the manuscript will be incorporated. Introduction: The authors showed the high prevalence of Musculoskeletal disorder (MSD) among dental health care providers in a worldwide scenario. MSD-related problems seem to be a significant issue for this profession and this is an already known problem. The authors tried to put up possibly all available publications related to MSD and did this attempt. However, certain changes in the manuscript and corrections to spelling mistakes or typos are recommended. The exact definition of MSD is missing. Response: MSDs are defined as musculoskeletal system and connective tissue diseases and disorders when the event or exposure leading to the case is bodily reaction (e.g., bending, climbing, crawling, reaching, twisting), overexertion, or repetitive motion. MSDs do not include disorders caused by slips, trips, falls, or similar incidents (Bureau of Labor Statistics of the Department of Labor. NIOSH workers health chartbook 2004. NIOSH Publication No. 2004-146. Washington, D.C). We will add the same into the mansucript. Methods: 1) Inclusion Criteria: Question: Is there any previously published systematic review included in this study? Response: No Question: Hygienists mentioned here are missing in Table 2. Are they included in auxiliaries? Response: Yes Question: What kind of studies were included - Cross-sectional/cohort/case-control? Response: We included studies from which prevalence could be calculated. However, most of the included studies were cross-sectional (n=86) Question: Were there any self-reported studies? Response: All the studies were self-reported 2) RoB: Question: What is the cutoff point used in this study? It is not mentioned that some cut-off point was used here. Response: Each question has two levels, low risk (0) and high risk (1). The total of all nine questions was used to categorize the studies as “low (0–3), moderate (4–6), or high risk (7–9)”. (This information was in the risk of bias assessment in the methodology section) Question: How many articles were excluded after RoB calculations? Response: Nil. Sensitivity analysis did not show any change in the overall estimate 3) Results: Question: Meta-analysis was performed for 89? Or 88? Response: Meta-analysis was performed for 89 estimates that yielded from 88 publications. Question: What is the reason for the exclusion of 146 publications after the full screening of the full text? Response: During the full-text screening of the publications, we excluded studies which had unclear outcome (n=138), inappropriate study design (n=4), short communications (n=2), and secondary analysis (n=2). This information is presented in figure 1. 4) Prevalence: There is a repeated word “than than”. Response: Thank you. We will incorporate the change. 5) Geographic distribution: Question: The authors mentioned that Australia (4), Africa (1), and north and south America (7,4) why not Asia and Europe (N=?)? Response: We didn’t want to replicate the data that has been presented in tables. Continent and country wise estimates with number of studies were presented in table 2. It may be better to revise this “One study from Africa (N=1)\". Response: We will incorporate the change. 6) Figure 1: Question: Where is the N=89 in this chart? Response: 89 estimates were obtained from 88 publications. In the flow chart we have highlighted the process flow with respect to the number of publications. Question: What are the other sources used to identify additional records? Responses: We have sought additional publications from the reference list given at the end of each article. 7) Table 2: Question: Why is Africa not mentioned here? Response: As there was only one study reported from Africa, we could not pool the estimate. Question: Malaysia has the highest estimates. What is the reason? Is it due to the inappropriate definition of MSD in Malaysia? Response: We could not identify the reason for this. Many factors like the number of clinical working days/week, working hours/day, type and duration of procedures, specialization, number of patients/days, remedial measures, and history of MSD in the past can have substantially impact on the estimates of MSD. Question: The estimate for one year (0.82) is more than for a lifetime (0.78); What may be the possible reason? Response: Different studies have used different time frames to report the MSD. Most studies have reported one year prevalence (n=65) followed by lifetime prevalence. Some studies reported both. Hence, it is not possible to substantiate what could be reason of this disparity. There could be possibility of recall bias among the participants that report lifetime events when compared to one-year events. Question: What is the definition of “mixed”? Response: Studies that have not reported estimates separately for dentists, dental students or dental auxiliaries or studies that included different type of dental personnel. Question: Dental auxiliaries seem to have more difficulties with MSD with an estimate of 0.83 (0.69-0.97). Is it not because 2-3 categories are grouped together? Response: Among the ten publications that were included in the dental auxiliaries, 6 studies were among dental hygienists (prevalence: 80.42 to 98.36), two were among dental technicians (prevalence 23.8 and 100%) and one was among dental assistant (prevalence 81.71%) and one was among dental auxiliaries (prevalence 81.7). Hence, estimates are high and may not related to the categorisation. 8) Limitation: Question: It’s mentioned that “the exclusion of studies published in other languages”; does it mean other than English? Response: Yes Discussion: Question: What was the national survey estimate? Mentioned in references 37,80? Better to mention Greek and Czech surveys. Response: Yes, we will incorporate the change. Question: Reference 37, is it a national survey? Response: No. It is a study among dental personnel in US dental army. Question: What was the prevalence of the studies that used the Nordic questionnaire? Response: 45 studies used Nordic questionnaire."
}
]
},
{
"id": "151602",
"date": "29 Nov 2022",
"name": "Preethi Balan",
"expertise": [
"Reviewer Expertise Clinical oral health 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\nThe authors have studied the prevalence estimates of MSD among dental healthcare workers using a systematic search of five databases. The authors observed high prevalence of MSD among dental healthcare workers which highlights the importance of them becoming aware of and adopting appropriate ergonomic postures early in their careers in order to reduce work-related MSD.\nThe paper is well written and covers most of the aspects in this issue. There are a few suggestions below:\n\nWere the two reviewers calibrated before doing the search? If possible the authors can provide the kappa statistics for agreement between the two reviewers.\n\nThe authors can provide the numbers of hits with each of the five databases in the prisma flowchart.\n\nThe authors can provide the years included in the search. Also if there was any manual search carried out in addition to the automated search.\n\nPlease elaborate in the inclusion/exclusion criteria regarding the type of studies considered, e.g. randomized and controlled clinical trials/observational studies/case control studies etc.\n\nAre the rationale for, and objectives of, the Systematic Review clearly stated? Yes\n\nAre sufficient details of the methods and analysis provided to allow replication by others? Partly\n\nIs the statistical analysis and its interpretation appropriate? Yes\n\nAre the conclusions drawn adequately supported by the results presented in the review? Yes",
"responses": [
{
"c_id": "9101",
"date": "05 Jan 2023",
"name": "Kalyana Pentapati",
"role": "Author Response",
"response": "Thank you for your time and efforts to improve the manuscript. Below are the responses: Query: Were the two reviewers calibrated before doing the search? If possible the authors can provide the kappa statistics for agreement between the two reviewers. Response: Yes, reviewers were calibrated before doing the screening. Agreement between the reviewers for title and abstract screening and full text screening showed almost perfect agreement (Kappa: 0.94 and 0.98 respectively). We have added this information in the manuscript. Query: The authors can provide the numbers of hits with each of the five databases in the prisma flowchart. Response: Yes, this information is there in the manuscript. We will add this in the flowchart also. A comprehensive systematic search of five databases (Scopus (1080), Embase (592), CINAHL (728), Web of Science (514), Dentistry & Oral Sciences Source (750)) yielded a total of 3664 articles. Query: The authors can provide the years included in the search. Also if there was any manual search carried out in addition to the automated search. Response: Search was performed from inception to 5 August 2021. No limits were applied. We have sought additional publications from the reference list given at the end of each article. Query: Please elaborate in the inclusion/exclusion criteria regarding the type of studies considered, e.g. randomized and controlled clinical trials/observational studies/case control studies etc. Response: Cross-sectional studies and cohort studies, where prevalence data can be extracted or calculated were included. We have added this information in the manuscript."
}
]
}
] | 1
|
https://f1000research.com/articles/11-1062
|
https://f1000research.com/articles/10-266/v1
|
01 Apr 21
|
{
"type": "Research Article",
"title": "Comparison of gender, age, and body mass index for spatiotemporal parameters of bilateral gait pattern",
"authors": [
"Turki Abualait",
"Mohammad Ahsan",
"Turki Abualait"
],
"abstract": "Background: Studies on the gaits parameters have been identified on the patient population. Most researchers confirm that the patients walk differently than normal people and they may have a greater risk of falls. Consistent finding and description of gender, age, and body mass index differences in gait studies is rare in healthy subjects. This research was performed to compare spatiotemporal parameters of gait between gender, as per their age and body mass index level.\n\nMethods: A cross-sectional study was conducted with forty-five young adults (F=20, M=25). Stadiometer and Physilog 4 inertial sensors were used for data collection. A gait analyzer 5.2 software (GaitUp, S.A. Lausanne, Switzerland) was used to determine spatiotemporal parameters.\n\nResults: No statistically significant differences were found in any bilateral foot gait parameters with respect to gender, age, and body mass index. Females were found with higher total double support and cadence than males. Cadence also increases with age. Obese people showed lower gait speed, cadence, and total double support. Conclusion: These findings may be beneficial to those who have abnormal gait pattern due to age, body mass index differences, decreased muscle strength, spasticity, and joint mobility. This important information should be considered to rehabilitate patients with abnormal gait patterns to controlling dynamic balance and risk of falling.",
"keywords": [
"Spatiotemporal Parameters",
"Gait",
"GaitUp",
"Gender",
"Age",
"Body Mass Index"
],
"content": "Introduction\n\nSpatiotemporal is the primary measurement in gait analysis. There is a common perception that males walk differently from females. The differences may occur due to physical capacities such as muscular strength, endurance, coordination, flexibility, agility, and emotional balance. Walking patterns may also fluctuate by age, body mass index (BMI), surface, course of time, and changes from stride to stride. Gender differences in a healthy population reveal contradictory discoveries regarding spatiotemporal parameters of gait. Stride-to-stride variability has been attributed to the underlying mechanism that produces human gait in healthy people.1\n\nMany studies have either been surprisingly limited in their investigation, contradictory, or equivocal between genders. One study reported that stride characteristics do not have any gender differences during walking.2 Another study revealed that during walking, the speed is the same in males and females, but that the step length is shorter in females.3 On the other hand, a study reported males to walk faster than females, and that female step length is shorter than males.4 A previous study concluded that healthy older females walk with shorter stride length and higher cadence when compared to males.5 Females have been indicated to walk with lower preferred speed, smaller step length, but increased cadence compared with males.6 It has also been reported that healthy females had a higher velocity and bigger cadence, swing phase, stride length, and single support phase and a lower double support phase and stance phase compared with gait disorder.7 Heredia-Jimenez and Orantes-Gonzalez reported that while dealing with healthy females and females with fibromyalgia, there were significant differences in stride length, velocity, swing time variability, cadence, and stance gait.8 Several pieces of research also concluded that mean stride times can be affected by gender, whereas other temporal parameters such as stance, swing, double support times, and temporal variability unchanged in healthy young adults and older people.2,9\n\nAdvance aging factors affect gait pattern. Many studies have been reported that gait ability declines with age. A prior study found that age differences are connected with slower gait speed, shorter stride length, and wider stride width.10 Compared to young adults, the elderly adult gait model is characterized by shorter step length, slow gait speed, and a reduced range of motion at the hip.11 A study showed that when walking on a compliant surface, young and older people increase cadence and reduce velocity.12 One of the most consistent age-related changes has been shown as a decline in gait speed.13 There are many fall injuries associated with increasing age, from young adults to middle-aged adults to older adults, as the aging process is accompanied by changes in body composition.14 Obesity is a primary risk factor for many diseases which also negatively affects physical functioning, especially walking ability and performance.15 The effects of obesity and overweight on gait parameters in adults not well known. In obese adults, gait is distinguished by slow step frequency, shorter step length, longer stance phase, and walking speed is minimized.14 Stride frequency and stride length did not differ between moderately obese individuals and healthy weight individuals.16 Obese individuals walk slowly with a shorter step length than underweight individuals.17 There is a lack of knowledge on spatiotemporal parameters from the underweight, healthy, overweight, and obese population.\n\nMany methods were suggested to investigate the spatiotemporal patterns of the gait sequence to understand the differences between males and females. Thang et al. used only the smartphone's accelerometer sensor to user authentication while Zong and Deng captured walking information from both the accelerometer and gyroscope sensors.18,19 Recently, the Physiolog gait analysis system from GaitUp (SA, Lausanne, Switzerland) has become a popular and is an important tool for the objective evaluation and planning of rehabilitation strategies for an abnormal gait pattern. The use of these sensors has been described previously, and they demonstrate good accuracy and perception of gait analysis.20,21 Despite this evidence, a limited number of studies have investigated the spatiotemporal parameters of gait using internal wearable sensors.\n\nTherefore, the purpose of this current study was to compare the characteristics of spatiotemporal gait based on gender, age, and BMI level. In particular, the aim of the study is to answer the question of whether there are differences between gait parameters. To achieve the objective of this study for gait parameters in gender, age, and BMI level, we tried a statistical comparison between males and females, age differences, and BMI categories for spatiotemporal gait analysis.\n\n\nMethods\n\nA cross-sectional study design was chosen to achieve the objective of this study. This study was conducted in accordance with the Declaration of the Principles of Helsinki.\n\nThis study was approved by the local Institutional Review Board at Imam Abdulrahman Bin Faisal University with IRB-Number: IRB-2019-03-255.\n\nSample size was calculated based on the calculation formula for similar type of study.22 The standard normal variate (Z value) was set at a significance level of 5% with (α = Zα = 1.960, β = Zβ = .842) and C = 0.5 x in [(1 + r)/(1 − r)] = .375) based on related previous studies (Lewek et al., 2014b).23 The required sample size was estimated to be 39 with adding an expected drop out of 20%; thus, the sample size was projected to be 45 subjects.\n\n45 young adults (25 male and 20 female) were included in this study. Their mean (standard deviation [SD]) age, height, weight, and BMI were 21.82(3.93) years, 165.83(8.00) cm, 66.10(13.19) kg, and 24(3.89) respectively. The study was carried out from June to August of 2019, data were collected during single session for each participant at biomechanics gait lab in Imam Abdulrahman Bin Faisal University. All participants who met the inclusion criteria had no history of musculoskeletal or neurological deficits which could affect their gait performance, and all were able to understand and follow commands. Exclusion criteria were any significant gait-associated impairments or any previous injury that has an effect on gait performance, psychiatric illness and severe cognitive deficits. All participants wear flat shoes during taking the assessment to standardize the procedure of the test and minimize the effects of any footwear on gait performance. No bias were identified that would affect this study.\n\nTable 1 showed significant differences in height (p = .000) and weight (p = .001) for male and female participants. Age, leg length, and BMI level showed insignificant differences for both genders.\n\n\nEquipment\n\nWeight, height, and BMI were measured with a portable electronic calibrated scale (Detecto Scale-model 750, USA). Participants were asked to wear light clothing and take off their shoes for accurate measurement.\n\nTo measure spatiotemporal parameters of all participants Physilog 4 silver 10D from GaitUp (S.A., Lausanne, Switzerland) was used. Physilog has good accuracy and precision for gait analyses.20,21\n\nA total of 45 participants agreed to participate in the study. There were 20 female and 25 male participants. Before the actual test, all basic instructions were explained to the participants. After collecting their anthropometric data with a Detecto scale, physilog inertial sensors were placed on each participant's foot for the gait test. Participants were asked to walk at a comfortable pace for themselves, along a 10-meter straight path. The three trails were recorded. The average of the three trials was used for further analysis. In this study, the spatiotemporal parameters chosen for the analysis were gait speed, gait cycle, foot speed, stride length, total double support, and cadence for both legs. A gait analyser 5.2 software (GaitUp, S.A. Lausanne, Switzerland) was used to determine Gait Speed (meter/second), Gait Cycle (seconds), Foot Speed (meter/second), and stride length (meter), Total Double Support (% Cycle), and Cadence (Step/Min).\n\n\nStatistical analysis\n\nStatistical analysis was performed using IBM SPSS for windows, version-21 (IBM Crop. USA). Prior to analysis, data were screened for missing values and outliers. Descriptive analyses were conducted for anthropometric characteristics. Independent sample t-tests and one-way analysis of the variance (ANOVA) test was extended to find out the differences between different types of spatiotemporal parameters of gait for gender, age, and BMI levels. The significance level was set at the 0.05 level.\n\n\nResults\n\nThe results of the gait parameters for males and females' scores are presented in Table 2. When considering the mean score, males have higher gait speed, gait cycle, foot speed, and stride length than females, while females have higher total double support and cadence than males. Independent sample t-tests were conducted to compare the left and right foot’s gait speed, gait cycle, foot speed, stride length, total double support, and cadence for the males and females. There were no significant differences found in any gait parameters in the left and right foot for male and female participants.\n\nThe result of gait parameters based on age categories (18-25 and 30-38 years) scores are presented in Table 3. When considering the mean scores, the participants belonging to the 18-25 age category showed higher gait speed, gait cycle, foot speed, stride length, and total double support than participants belonging to the 30-38 age category. While the participants belonging to the 30-38 age categories showed higher left and right foot’s cadence than the participants belong to the 18-25 age category. Independent sample t-tests were conducted to compare the left and right foot’s gait speed, gait cycle, foot speed, stride length, total double support, and cadence for the age categories. The results showed that there were no significant differences found among any gait parameters in left and right foot for either age category.\n\nThe result of gait parameters based on their BMI levels (underweight, healthy, overweight, and obese) scores are presented in Table 4. When considering the mean scores, the participants according to their BMI level showed that underweight participants have the highest gait speed, foot speed, stride length, and cadence than the participants belong to other BMI categories. While the gait cycle and total double support for the left and the right foot are smaller than the other BMI categories participants. The participants belonging to the healthy BMI category show that the gait speed, foot speed, stride length, and cadence are higher, and their gait cycle is smaller than overweight and obese participants, while total double support for the left and the right foot is lower than overweight and higher than obese participants. The overweight participants showed the gait speed, gait cycle, foot speed, and the stride length are lower than obese participants, while total double support and cadence for left and right were higher for overweight participants than obese participants. The obese participants showed gait speed and cadence are lower and the gait cycle is higher than other BMI categories participants, foot speed is higher than healthy and overweight participants while lower than underweight participants. The total double support was lower than healthy and overweight participants but higher than underweight participants. Additionally, one-way ANOVA was conducted to compare the gait parameters among different BMI categories participants. The results showed that there were no significant differences in any gait parameters for the left and right foot for any BMI categories’ participants.\n\n\nDiscussion\n\nThe findings of this study indicated that gait speed, gait cycle, foot speed, and stride length were overall higher in male than female participants. Whereas cadence and double support were higher in female than male participants. The results suggest that there was no significant difference in male and female participants for the left and right foot gait parameters. Most previous studies suggested that step length and cadence are responsible for the gait speed, and these measurements have some biological dependence on the height of the individual.24 Kerrigan et al have observed that healthy males who walked at the same walking speed as females showed lower cadence and longer step length than the female.25 It was reported that the healthy weight category women walk with reduced stride length and higher cadence with respect to men, in order to achieve comparable speed values.5 It was reported that the spatiotemporal gait parameters for both genders showed that females have greater stride time while males performed higher stride length, step time, cadence, and walking speed.26 Kerrigan et al revealed that there are few significant gender differences for spatiotemporal data, with a longer normalized stride length and greater cadence in females. Both genders had the same step width and walking velocity due to the effort that females made to increase their stride length with the aim of walking as fast as males.25 We found that males and females did not differ significantly in the spatiotemporal parameters of normal gait speed, gait cycle, normal stride length, and cadence. These findings are in partial agreement with the findings of the above-mentioned studies. Gender differences may also be associated with body proportions between males and females. Muscle strength and bone configuration may have importance to determine gait parameters outcomes between genders.\n\nThe results of gait parameters based on two age categories (18-25 and 30-38 years old) scores are presented in Table 3. The participants belonging to the 18-25 age category showed higher gait speed, gait cycle, foot speed, stride length, and total double support than participants belonging to the 30-38 age category. However, the participants belonging to the 30-38 age categories showed higher left and right foot’s cadence than the participants belonging to the 18-25 age category. The results of the present study indicated that there is no significant differences in between any spatiotemporal parameters in the age groups. Several studies indicated that these spatiotemporal measures deteriorate more rapidly with age for women than for men,27 while others found no interactions between the sexes during aging.28 Results from Frimenkoa et al study indicated that there was a significant difference in both genders which slowed their gait speed with age. At a similar age, females have a higher cadence and smaller stride lengths than males. As the age increases, gait speed decreases in both genders, while females maintain smaller step length and higher cadence.29 Moreover, Abreu and colleagues uncovered a negative relationship between aging and stride length during gait due to increased eccentric activity of the quadriceps muscles during the final stage of double support or increased eccentric activity in the hamstrings during the final balance phase that occurs with increasing age.30 Older adults reduce their gait speed and take shorter steps while increasing the time of double support to maintain their dynamic balance.31 The results of the present study also support previous findings that age-related changes in gait speed through shorter steps were adopted for a safer and steadier gait.\n\nOur findings showed that the underweight participants have the highest gait speed, foot speed, stride length, and cadence than the participants belong to other BMI categories, while gait cycle and total double support for the left and the right foot are smallest than the other BMI categories participants. The participants belonging to the healthy weight category showed that the gait speed, foot speed, stride length, and cadence are higher, and the gait cycle is smaller than the overweight and obese category participants. However, their total double support for the left and the right foot is lower than the overweight category participants and higher than the obese category participants. The overweight category participants demonstrated that the gait speed, gait cycle, foot speed, and the straight length is lower than the obese category participants, while total double support and cadence for left and right showed higher for the overweight category than the obese category participants. The obese category participants showed gait speed and cadence are lower and the gait cycle is higher than other BMI categories participants, foot speed is higher than the healthy weight and overweight participants while lower than the underweight participants. The total double support was lower than the healthy weight and overweight categories but higher than the underweight category participants.\n\nPeople who are overweight and/or obese are known to have a functional implication in everyday life. It has been shown that excess weight alters the normal gait mechanism.16 Our findings are in line with previous research, with obese adults walking with shorter strides in length, large stride width, and shorter stride length compared to healthy weight adults when walking at a self-defined speed.32 On the other hand, when comparing healthy weight and obese adults, no differences were found in step length.16–17 However, these results may be directly due to the effect of speed.16 It has been clearly indicated in the literature that obese people tend to have reduced stride length, swing phase duration, cadence, walking speed, increased stance phase, step width, and double support.33 A recent review summarizing the results of 25 studies on the gait of obese children34 concluded that there is moderate evidence of increased step width and stance phase duration, while for all other spatiotemporal parameters, the differences are either non-significant or inconsistent as our results suggest.\n\nSome limitations should be considered which might limit the generalizability of the findings. First, all participants were adults; different ages and weights were not involved in the study. Second, Physilog (GaitUp) is not a common device for determining spatiotemporal gait parameters, however this may be employed more in future research. Finally, no comparable group with gait disturbances was enrolled in this study to compare the spatiotemporal gait parameters with healthy subjects. Thus, future studies should compare the spatiotemporal parameters of gait between gender, as per their age and body mass index level between healthy adults and patient groups.\n\n\nConclusions\n\nIn summary, we quantified the spatiotemporal parameters of gait differences as per gender, age, and BMI levels. Our result suggests that there are differences in all the spatiotemporal parameters for gender, age, and BMI levels at the left and right foot, but these differences are not statistically significant to each other. These findings may be beneficial to those who have abnormal gait pattern due to age, BMI differences, decreased muscle strength, spasticity, and joint mobility. This important information should be considered when rehabilitating patients with abnormal gait patterns with to controlling dynamic balance and risks of falling.\n\n\nData availability\n\nHarvard Dataverse: “Comparison of Gender, Age, and Body Mass Index for Spatiotemporal Parameters of Bilateral Gait Pattern”, https://doi.org/10.7910/DVN/10WUSP.36\n\nThis project contains the following underlying data:\n\n• Raw data excel file\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\nConsent statement\n\nParticipants who agreed to participate in the study voluntarily were given a detailed written and verbal explanation of the study then asked to sign a written consent form.",
"appendix": "Acknowledgements\n\nThe authors would like to thank the Dean and Head of the Department of Physical Therapy, College of Applied Medical Sciences, Imam Abdulrahman Bin Faisal University for giving their permission to conduct this research. Besides that, the authors would like to express gratitude to all students and staff who participated in this study.\n\n\nReferences\n\nStergiou N, Moraiti C, Glakas G, et al.: The effect of walking speed on the stability of the anterior cruciate ligament deficient knee. Cli Biomech. 2004; 19: 57–63. PubMed Abstract | Publisher Full Text\n\nBarrett R, Noordegraaf MV, Morrison S: Gender difference in the variability of lower extremity kinematics during treadmill locomotion. J Mot Behav. 2008; 40: 62–70. PubMed Abstract | Publisher Full Text\n\nLaufer Y: Age and gender related changes in the temporal-spatial characteristics of forwards and backwards gaits. Physiother Res Int. 2003; 8: 131–142. PubMed Abstract | Publisher Full Text\n\nSamson MM, Crowe A, de Vreede PL, et al.: Differences in gait parameters at a preferred waking speed in healthy subjects due to age, height and body weight. Aging. 2001; 13: 16–21. PubMed Abstract | Publisher Full Text\n\nKo SU, Tolea MI, Hausdorff JM, et al.: Sex-specific differences in gait patterns of healthy older adults: results from the Baltimore Longitudinal Study of Aging. J Biomech. 2011; 44: 1974–1979. PubMed Abstract | Publisher Full Text | Free Full Text\n\nBohannon RW, Andrews AW: Normal walking speed: a descriptive meta-analysis. Physiotherapy. 2011; 97: 182–189. PubMed Abstract | Publisher Full Text\n\nHeredia JM, Garcia-Molina V, Porres Foulquie JM, et al.: Spatial-temporal parameters of gait in women with fibromyalgia. Clin Rheumatol. 2009; 28: 595–598. PubMed Abstract | Publisher Full Text\n\nHeredia-Jimenez J, Orantes-Gonzalez E: Gender differences in patients with fibromyalgia: a gait analysis. Clin Rheumatol. 2019; 38: 513–522. PubMed Abstract | Publisher Full Text\n\nHollman JH, McDade EM, Petersen RC: Normative spatiotemporal gait parameters in older adults. Gait Posture. 2011; 34: 111–118. PubMed Abstract | Publisher Full Text | Free Full Text\n\nKo SU, Stenholm S, Ferrucci L: Characteristic gait patterns in older adults with obesity - Results from the baltimore longitudinal study of aging. J Biomech. 2010; 43: 1104–1110. PubMed Abstract | Publisher Full Text | Free Full Text\n\nKo SU, Hausdorff JM, Ferrucci L: Age-associated differences in the gait pattern changes of older adults during fast-speed and fatigue conditions: results from the Baltimore longitudinal study of ageing. Age Ageing. 2010; 39: 688–694. PubMed Abstract | Publisher Full Text | Free Full Text\n\nRogers HL, Cromwell RL, Grady JL: Adaptive changes in gait of older and younger adults as responses to challenges to dynamic balance. J Aging Phys Act. 2008; 16: 85–96. PubMed Abstract | Publisher Full Text\n\nWinter DA, Patla AE, Frank JS, et al.: Biomechanical walking pattern changes in the fit and healthy elderly. Phys Ther. 1990; 70: 340–347. PubMed Abstract | Publisher Full Text\n\nRoubenoff R: Sarcopenia: Effects on Body Composition and Function. J Gerontol A Biol Sci Med Sri. 2003; 58: 1012–1017. PubMed Abstract | Publisher Full Text\n\nStenholm S, Sainio P, Rantanen T, et al.: Effect of co-morbidity on the association of high body mass index with walking limitation among men and women aged 55 years and older. Aging Clin Exp Res. 2007; 19: 277–283. PubMed Abstract | Publisher Full Text\n\nBrowning RC, Kram R: Effects of obesity on the biomechanics of walking at different speeds. Med Sci Sports Exerc. 2007; 39: 1632–1641. PubMed Abstract | Publisher Full Text\n\nDeVita P, Hortobagyi T: Obesity is not associated with increased knee joint torque and power during level walking. J Biomech. 2003; 36: 1355–1362. PubMed Abstract | Publisher Full Text\n\nThang HM, Viet VQ, Thuc ND, et al.: Gait identification using accelerometer on mobile phone. International Conference on Control, Automation and Information Sciences (ICCAIS). 2012; 344–348. Publisher Full Text\n\nZhong Y, Deng Y: Sensor orientation invariant mobile gait biometrics. IEEE International Joint Conference on Biometrics (IJCB). 2014: 1–8. Publisher Full Text\n\nBregou Bourgeois A, Mariani B, Aminian K, et al.: Spatio-temporal gait analysis in children with cerebral palsy using, foot-worn inertial sensors. Gait Posture. 2014; 39: 436–442. PubMed Abstract | Publisher Full Text\n\nDadashi F, et al.: Gait and foot clearance parameters obtained using shoe-worn inertial sensors in a large-population sample of older adults. Sensors. 2013; 14: 443–457. PubMed Abstract | Publisher Full Text | Free Full Text\n\nhttp\n\nLewek MD, Bradley CE, Wutzke CJ, et al.: The relationship between spatiotemporal gait asymmetry and balance in individuals with chronic stroke. J Appli Biomec. 2014; 30: 31–36. PubMed Abstract | Publisher Full Text\n\nHof AL: Scaling gait data to body size. Gait Posture. 1996; 4: 222–223. Publisher Full Text\n\nKerrigan DC, Todd MK, Croc UD: Gender differences in joint biomechanics during walking: normative study in young adults. Am. J. Phys. Med. Rehabil. 1998; 77: 2–7. PubMed Abstract | Publisher Full Text\n\nZakaria NK, Jailani N, Tahir NM: Gender differences in gait features of healthy children. Jurnal Teknologi (Sciences & Engineering). 2015; 77: 1–6. Publisher Full Text\n\nDoyo W, Kozakai R, Kim HY, et al.: Spatiotemporalcomponents of the 3-D gait analysis of community-dwelling middle-aged and elderly Japanese: age-and sex-related differences. Geriatr Gerontol Int. 2011; 11: 39–49. PubMed Abstract | Publisher Full Text\n\nVerlinden V, van der Geest J, Hoogendam YY, et al.: Gait patterns in a community-dwelling population aged 50 years and older. Gait Posture. 2013; 37: 500–505. PubMed Abstract | Publisher Full Text\n\nFrimenkoa R, Goodyeara C, Brueningb D: Interactions of sex and aging on spatiotemporal metrics innon-pathological gait: A descriptive meta-analysis. Physiotherapy. 2015; 101: 266–272. PubMed Abstract | Publisher Full Text\n\nAbreu SSE, Caldas CP: Gait speed, balance and age: A correlational study among elderly women with and without participation in a therapeutic exercise program. Revista Brasileira de Fisioterapia. 2008; 12: 324–330. Publisher Full Text\n\nPrince F, Corriveau H, Winter DHR: Gait in the elderly. Gait Posture. 1997; 5: 128–135. Publisher Full Text\n\nRunhaar J, Koes BW, Clockaerts S, et al.: A systematic review on changed biomechanics of lower extremities in obese individuals: A possible role in development of osteoarthritis. Obes. Rev. 2011; 12: 1071–1082. PubMed Abstract | Publisher Full Text\n\nWearing SC, Hennig EM, Byrne NM, et al.: The biomechanics of restricted movement in adult obesity. Obes. Rev. 2006; 7: 13–24. PubMed Abstract | Publisher Full Text\n\nMolina-Garcia P, Migueles JH, Cadenas-Sanchez C, et al.: A systematic review on biomechanical characteristics of walking in children and adolescents with overweight/obesity: Possible implications for the development of musculoskeletal disorders. Obes Rev. 2019; 20: 1033–1044. PubMed Abstract | Publisher Full Text\n\nAhsan M: “Comparison of Gender, Age, and Body Mass Index for Spatiotemporal Parameters of Bilateral Gait Pattern”. Harvard Dataverse, V1. 2021. Publisher Full Text\n\nAhsan M: “Comparison of Gender, Age, and Body Mass Index for Spatiotemporal Parameters of Bilateral Gait Pattern”. Harvard Dataverse, V1, UNF:6:qEkm00O3MsnfZ3ZGzAwpoQ== [fileUNF]. 2021. Publisher Full Text"
}
|
[
{
"id": "82919",
"date": "17 Aug 2021",
"name": "Moazzam Hussain Khan",
"expertise": [
"Reviewer Expertise Exercise physiology",
"Sports Injury",
"Chronobiology",
"biomechanics"
],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nPlease provide more information on the following:\nHow were the participants recruited for this study?\n\nNot enough detail regarding Physiolog 4 - please expand on this.\n\nReliability and validity of the instruments used.\n\nIn the procedure: the Detecto scale should be replaced with a stadiometer cum weighing scale.\n\nProvide full details for reference number 22.\n\nWhat about flat feet - have you included this in the study?\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? 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": "9094",
"date": "09 Dec 2022",
"name": "Mohammad Ahsan",
"role": "Author Response",
"response": "Please provide more information on the following: How were the participants recruited for this study? Participants were recruited through an open advertisement within the university campus. Not enough detail regarding Physiolog 4 - please expand on this. Physilog is a high-quality 3D accelerometer, 3D gyroscope and barometric pressure sensor. It provided raw 3D acceleration data at a sample rate of 128 Hz. It has an inbuilt SD memory card to store recorded data. Physilog application for gait was installed on a tablet/computer and connected with sensors. Stored data were transferred to the computer through the USB cable. Reliability and validity of the instruments used. Reference # 21,22. In the procedure: the Detecto scale should be replaced with a stadiometer cum weighing scale. Replaced. Provide full details for reference number 22. Provided. What about flat feet - have you included this in the study? No, we did not determine flat feet in this study. Our focus was on the gait pattern."
}
]
},
{
"id": "151494",
"date": "11 Oct 2022",
"name": "Haruhiko Sato",
"expertise": [
"Reviewer Expertise Physiotherapy"
],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nI read with interest this research article that addressed the comparison of gender, age, and body mass index on spatiotemporal parameters of gait. My main concern is that the authors stated the conclusion not standing on their statistical results. In the statistical results of this study, there was no difference between gender to spatiotemporal parameters of gait, however, the authors seem to conclude gender differences exist as like the previous related articles.\nThe following provides more specific comments by section:\nAbstract Results: The authors stated, \"Females were found with higher total double support and cadence than males.\" but this is not supported by the statistically significant result.\nConclusion: It's unclear what refers to \"these findings\". It refers to \"no differences for gender”? or refers to \"females showed higher cadence than males\"? The authors should state the conclusion more clearly and it should be based on the statistical results.\nIntroduction In the first paragraph, line 5: I think the authors should distinguish spatiotemporal parameters of gait (i.e., stride length or cycle time) and stride-to-stride variability parameters of gait (i.e., coefficient of variation for stride length or cycle time). The authors did not show any data on the variability of gait, so this sentence had better remove.\n2nd paragraph: Main focus of this paragraph was unknown. In the anterior part of this paragraph, the authors stated the gender difference in gait parameters, but in the posterior part of the paragraph, the difference between people who have a disorder and people who did not have a disorder. With the various topic included in one paragraph, it comes difficult to understand what the authors want to discuss here, and why the authors try to compare gender differences in spatiotemporal parameters of gait.\n3rd paragraph: Again, two topics are mixed. The former part is about gait and age, and the latter part is about gait and obesity. I would recommend the authors write a paragraph each topic separately.\nMethod Sample size: Regarding the sample size calculation, isn't the calculation shown here for the sample size calculation for the correlation analysis? Since this study mainly compared gait parameters between men and women, I wonder if the authors would be used the expected standard deviation of the gait parameters for the sample size calculation.\nEquipment More explanation is needed about the measurement using the Physiology 4. What is the measurement frequency? Where and how is the sensor attached? What kind of gait parameters are obtained? etc.\nProcedure I do not understand what \"Foot speed\" means. Please explain it in more detail. In addition, the gait parameters are usually expressed as the combined value of both sides of the legs, especially in gait speed and cadence. The authors had better show them.\nStatistical analysis The authors are comparing groups by age and BMI, but the criteria for dividing into groups were unknown. Please clarify how the group was made.\nResults Please delete the sentence like \"Independent sample t-tests were conducted to compare...\". It should be stated in the statistical analysis section of the methods.\nIn tables 2 to 4 please show the number of samples by gender, age, or BMI group.\nDiscussion In the first paragraph, lines 1-2: I don't think it can be considered a major result because it is not a statistically significant result. The authors should discuss statistically significant results first. Also, in the second and third paragraphs, the authors state insignificant results first.\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? No",
"responses": [
{
"c_id": "9095",
"date": "05 Dec 2022",
"name": "Mohammad Ahsan",
"role": "Author Response",
"response": "The following provides more specific comments by section: Abstract Results: The authors stated, \"Females were found with higher total double support and cadence than males.\" but this is not supported by the statistically significant result. Changed Conclusion: It's unclear what refers to \"these findings\". It refers to \"no differences for gender”? or refers to \"females showed higher cadence than males\"? The authors should state the conclusion more clearly and it should be based on the statistical results. Changed Introduction In the first paragraph, line 5: I think the authors should distinguish spatiotemporal parameters of gait (i.e., stride length or cycle time) and stride-to-stride variability parameters of gait (i.e., coefficient of variation for stride length or cycle time). The authors did not show any data on the variability of gait, so this sentence had better remove. Removed. 2nd paragraph: Main focus of this paragraph was unknown. In the anterior part of this paragraph, the authors stated the gender difference in gait parameters, but in the posterior part of the paragraph, the difference between people who have a disorder and people who did not have a disorder. With the various topic included in one paragraph, it comes difficult to understand what the authors want to discuss here, and why the authors try to compare gender differences in spatiotemporal parameters of gait. Author wants to highlight the previous differences among gender for gait parameters. 3rd paragraph: Again, two topics are mixed. The former part is about gait and age, and the latter part is about gait and obesity. I would recommend the authors write a paragraph each topic separately. Author separates both paragraphs, one for age effect on gait and another one is for body composition. Method Sample size: Regarding the sample size calculation, isn't the calculation shown here for the sample size calculation for the correlation analysis? Since this study mainly compared gait parameters between men and women, I wonder if the authors would be used the expected standard deviation of the gait parameters for the sample size calculation. Sample size calculation removed Equipment More explanation is needed about the measurement using the Physiology 4. What is the measurement frequency? Where and how is the sensor attached? What kind of gait parameters are obtained? etc. Information added Procedure I do not understand what \"Foot speed\" means. Please explain it in more detail. Separate foot (left and right) speed. In addition, the gait parameters are usually expressed as the combined value of both sides of the legs, especially in gait speed and cadence. Yes, It's true; we measure all the mentioned parameters for both feet. All the outcome measures are according to the software outcome. For references: https://www.youtube.com/watch?v=2jy7m6RxC9Q The authors had better show them. Statistical analysis The authors compare groups by age and BMI, but the criteria for dividing into groups were unknown. Please clarify how the group was made. The age criteria were mentioned in table 3 as age range (18-25) and (30-38) The Body Mass Index (BMI) groups were classified according to the universal criteria Underweight (Less than 18.5), Healthy (18.5-24.9), Overweight (25-29.9), and Obese (Higher than 30). Results Please delete the sentence like \"Independent sample t-tests were conducted to compare...\". It should be stated in the statistical analysis section of the methods. Deleted In tables 2 to 4 please show the number of samples by gender, age, or BMI group. Added Discussion In the first paragraph, lines 1-2: I don't think it can be considered a major result because it is not statistically significant. The authors should discuss statistically significant results first. Also, in the second and third paragraphs, the authors state insignificant results first. Changed accordingly"
}
]
},
{
"id": "153604",
"date": "31 Oct 2022",
"name": "Hanatsu Nagano",
"expertise": [
"Reviewer Expertise Gait biomechanics",
"falls prevention",
"ergonomics",
"robotics",
"rehabilitation"
],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nGait characteristics contain various health information and spatio-temporal parameters have been recognised as the fundamental descriptions of individuals’ walking patterns. The current research investigated a total of 45 healthy young adults’ spatio-temporal parameters sub-classified by gender, age and body mass index. While the study was the important topic, there are some fundamental concerns to be clarified.\nFirst, the study seemed to investigate effects of (i) gender, (ii) age and (iii) body mass index on spatio-temporal gait parameters. However, the total sample is only 45 and there seemed to be only the healthy young population included in the study. This limited sample may not be sufficient in leading any meaningful conclusion about age/body mass index effects on spatio-temporal gait parameters. There are the previous studies that have incorporated much larger samples about spatio-temporal parameters; therefore, the current study is expected to demonstrate the unique findings.\nSecond, the introduction can be improved by elaborating on the three separate discussions about (i) gender, (ii) age and (iii) BMI effects on gait.\nThird, I wonder if there were any inclusion criteria in the study in terms of age. Were participants recruited widely from the community (e.g., targeting participation of 65+ yrs population)? The sample population seemed to be limited to the healthy and young.\nFourth, some gait parameters were not defined properly. For example, what was foot speed? Was it the maximum speed during the swing phase, the average or something else? Similarly, what was Gait Cycle?\nFifth, tables 2 & 3 should show sample sizes based on the subclassifications (i.e., age, BMI).\nSixth, what was the importance of this study if no significant differences were identified in any way? Step length or other parameters may possibly reveal gender differences if normalisation techniques were employed.\n\nIs the work clearly and accurately presented and does it cite the current literature? No\n\nIs the study design appropriate and is the work technically sound? Partly\n\nAre sufficient details of methods and analysis provided to allow replication by others? No\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\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": "9096",
"date": "05 Dec 2022",
"name": "Mohammad Ahsan",
"role": "Author Response",
"response": "Gait characteristics contain various health information and spatio-temporal parameters have been recognised as the fundamental descriptions of individuals’ walking patterns. The current research investigated a total of 45 healthy young adults’ spatio-temporal parameters sub-classified by gender, age and body mass index. While the study was the important topic, there are some fundamental concerns to be clarified. First, the study seemed to investigate effects of (i) gender, (ii) age and (iii) body mass index on spatiotemporal gait parameters. However, the total sample is only 45 and there seemed to be only the healthy young population included in the study. This limited sample may not be sufficient in leading any meaningful conclusion about age/body mass index effects on spatiotemporal gait parameters. There are the previous studies that have incorporated much larger samples about spatiotemporal parameters; therefore, the current study is expected to demonstrate the unique findings. Second, the introduction can be improved by elaborating on the three separate discussions about (i) gender, (ii) age and (iii) BMI effects on gait. Paragraph # 2 elaborates the gender and gait; Paragraph # 3 is related to age differences in gait parameters; paragraph # 3 is on BMI or obesity's effect on gait. Third, I wonder if there were any inclusion criteria in the study in terms of age. Were participants recruited widely from the community (e.g., targeting participation of 65+ yrs population)? The sample population seemed to be limited to the healthy and young. To recruit the participant an open advertisement was done within the university campus. We got participants only age range 19-38 only. Fourth, some gait parameters were not defined properly. For example, what was foot speed? Was it the maximum speed during the swing phase, the average or something else? Similarly, what was Gait Cycle? Meaning defined added Fifth, tables 2 & 3 should show sample sizes based on the subclassifications (i.e., age, BMI). Added Sixth, what was the importance of this study if no significant differences were identified in any way? Step length or other parameters may possibly reveal gender differences if normalisation techniques were employed."
}
]
},
{
"id": "153602",
"date": "08 Nov 2022",
"name": "Hajra Masood",
"expertise": [
"Reviewer Expertise computer vision",
"machine learning",
"gait analysis."
],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe study describes the effects of gender, age and obesity on spatiotemporal parameters of gait.\nThe findings are backed with data and statistical analysis, but they lack logical discussion and a future direction for the research. The data and procedure of experiment is clearly mentioned but reproduction and verification of results is difficult as data collection is conducted on specialized equipment.\nThe inferences made after research are generalized in nature.\n\nIs the work 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": "9097",
"date": "05 Dec 2022",
"name": "Mohammad Ahsan",
"role": "Author Response",
"response": "The study describes the effects of gender, age and obesity on spatiotemporal parameters of gait. The findings are backed with data and statistical analysis, but they lack logical discussion and a future direction for the research. The data and procedure of experiment is clearly mentioned but reproduction and verification of results is difficult as data collection is conducted on specialized equipment. The inferences made after research are generalized. Most of the changes have been made."
}
]
}
] | 1
|
https://f1000research.com/articles/10-266
|
https://f1000research.com/articles/10-1111/v1
|
03 Nov 21
|
{
"type": "Research Article",
"title": "Leveraging biochemical reactions to unravel functional impacts of cancer somatic variants affecting protein interaction interfaces",
"authors": [
"Francesco Raimondi",
"Joshua G. Burkhart",
"Matthew J. Betts",
"Robert B. Russell",
"Guanming Wu",
"Joshua G. Burkhart",
"Matthew J. Betts",
"Robert B. Russell"
],
"abstract": "Background: Considering protein mutations in their biological context is essential for understanding their functional impact, interpretation of high-dimensional datasets and development of effective targeted therapies in personalized medicine. Methods: We combined the curated knowledge of biochemical reactions from Reactome with the analysis of interaction-mediating 3D interfaces from Mechismo. In addition, we provided a software tool for users to explore and browse the analysis results in a multi-scale perspective starting from pathways and reactions to protein-protein interactions and protein 3D structures. Results: We analyzed somatic mutations from TCGA, revealing several significantly impacted reactions and pathways in specific cancer types. We found examples of genes not yet listed as oncodrivers, whose rare mutations were predicted to affect cancer processes similarly to known oncodrivers. Some identified processes lack any known oncodrivers, which suggests potentially new cancer-related processes (e.g. complement cascade reactions). Furthermore, we found that mutations perturbing certain processes are significantly associated with distinct phenotypes (i.e. survival time) in specific cancer types (e.g. PIK3CA centered pathways in LGG and UCEC cancer types), suggesting the translational potential of our approach for patient stratification. Our analysis also uncovered several druggable processes (e.g. GPCR signalling pathways) containing enriched reactions, providing support for new off-label therapeutic options. Conclusions: In summary, we have established a multi-scale approach to study genetic variants based on protein-protein interaction 3D structures. Our approach is different from previously published studies in its focus on biochemical reactions and can be applied to other data types (e.g. post-translational modifications) collected for many types of disease.",
"keywords": [
"Structural bioinformatics",
"network bioinformatics",
"biological process",
"variant interpretation",
"biochemical reactions",
"PPI network",
"biological pathways",
"cancer mutations",
"Reactome"
],
"content": "Introduction\n\nSomatic mutations in cancer driving genes are a primary cause of oncogenesis. Large-scale cancer sequencing projects, including TCGA and ICGC, have uncovered many somatic mutations in cancer related genes.1,2 Though a plethora of studies have been published investigating the molecular mechanisms of mutations in these genes and how they cause cancer, detailed and comprehensive biochemical mechanisms remain undiscovered. While recent efforts have been devoted to development of integrated bioinformatics pipelines for the reliable prediction of cancer driver mutation,3 the mechanistic details from such predictions are usually not available, therefore hampering deeper biological interpretation and clinical translation.4 The majority of current approaches for these studies are at the gene or protein, protein-protein interaction or pathway levels. The gene-level analysis tells us what changes occur in genes or proteins, but not the functional impact, while the pathway-level analysis may reveal potential overall impact of mutations in cancer drivers, but not how. The protein-protein interaction level analysis may help us infer possible impact on the interactions of mutated proteins, but such analysis does not extend to the pathway context because a protein-protein interaction may be involved in multiple pathways. For example, an interaction between GAB1 and EGFR has been annotated by BioGrid (https://thebiogrid.org/108824/summary/homo-sapiens/gab1.html) and involved in both Signaling by EGFR (https://reactome.org/PathwayBrowser/#/R-HSA-177929) and Signaling by ERRB2 (https://reactome.org/PathwayBrowser/#/R-HSA-1227986) as annotated in Reactome via different reactions.\n\nSeveral studies have been published to survey cancer somatic variants using protein-protein interaction 3D structures (e.g. 5–10). However, these studies were all based on experimentally determined protein-protein 3D structures, which covers only 6% of currently known human protein-protein interactions based on the latest release of interactome3d (https://interactome3d.irbbarcelona.org/results.php?queryid=human11). To increase the coverage of 3D structures for protein-protein interactions, several groups (e.g.11–15) have employed homology modeling approaches using 3D structures of homologous proteins as templates. Even using homology modeling, the structural coverage of the human interactome remains quite low (12% in interactome3d). The Mechismo approach16 was developed to predict the functional consequences of variants and post-translational modifications (PTMs), such as phosphorylations and acetylations, at biomolecular 3D interfaces. The method extends predictions to proteins with no 3D structures as a function of sequence similarity to 3D templates assessed through BLAST alignments.17 This tool allows for inspecting positional effects of mutations and modifications for protein-protein, protein-nucleic acids and a subset of protein-chemical interactions which we previously demonstrated to be as effective as comparative modeling in identifying residues at interaction interfaces without intensive computation.\n\nPathway and network-based approaches are routinely used in cancer data analysis. Projecting perturbed genes in the contexts of pathways and networks provides a knowledge framework for researchers to understand the potential roles of genetic aberrations that cause cancer. Pathway and network knowledgebases provide the foundation to perform pathway and network-based analysis. Reactome18 is the most comprehensive open source biological pathway knowledgebase, covering nearly 60% of total human protein coding genes with annotation of over 13,200 complexes and 13,500 reactions that are organized into approximately 2,500 manually curated pathways (Release 75, December 2020). Pathways in Reactome are annotated as linked biochemical reactions, covering all types of biological processes, including metabolism, signal transduction, cell cycle, DNA replication and repair, programming cell death, immune system, development biology and cell-cell interaction.\n\nLeveraging the protein-protein 3D structural information generated from Mechismo and the rich set of biochemical reactions in Reactome pathways, we conducted a systematic analysis of protein structural variants in cancers using the somatic mutation data from TCGA. Our analysis is focused on functional impact of variants at 3D interfaces mediating biochemical reactions, striking a balance between the gene or protein level and pathway level analyses and unraveling novel biochemical insights of oncogenesis. In addition, we have developed a new set of features for Reactome’s Cytoscape app, ReactomeFIViz,19 enabling researchers to browse and explore results from this study.\n\n\nResults\n\nWe developed a robust analysis approach to analyze cancer somatic mutations in the context of protein-protein interactions, biological reactions, and pathways (Figure 1). We applied this approach to the TCGA dataset by first mapping somatic mutations to UniProt canonical protein sequences and then onto 3D structures of protein-protein interactions as we have done previously through Mechismo.20 The mapped protein-protein interactions are then traced back to reactions annotated in Reactome, which are tested for statistical significance. Significant pathways can be detected through similar statistical analysis (Methods) or may be found by mapping significant reactions to pathways that contain them. By combining the analysis results from protein-protein interactions, reactions and pathways, we draw biological insights.\n\nWe considered a total of 1.79 M non-synonymous somatic mutations from TCGA across 32 cancer types and 7120 unique samples carrying somatic mutations. These were mapped onto canonical UniProt sequences, yielding 1.16 M unique protein variants (Methods). Mechismo analysis revealed that 405 K mutations (22%) mapped to known 3D structures and 103 K (25%) of them were found at interaction interfaces, for a total of 6668 (59%) samples carrying at least one interface perturbing mutation (Figure 2A). The majority of mutations were found at protein-protein and protein-chemical interfaces (60 K and 48 K, respectively) while 14 K were predicted at protein-nucleic acid interfaces.\n\nCancers shown in rows were sorted based on total number of samples.\n\nTo define the biological processes affected by interface mutations, we mapped 3D interaction interfaces to Reactome’s Functional Interaction Network (FIN),21 involving both protein-protein and protein-chemicals interactions (Methods). We matched 6559 of 17225 (38%) FIN Protein-chemical interactions and 24363 of 183672 (13%) FIN PPIs to 3D interaction interfaces. We assessed the statistical significance of mutation enrichment at interaction interfaces through a random background model obtained by shuffling the same substitutions between equivalent positions in the same proteins (Extended data: Table S138; Methods). Of the 2974 (26%) samples affected by significantly perturbed interfaces, 82% of them (2437 samples) carried mutations at significant FIN interfaces and 32% (3694 samples) carried mutations mapping either to non-significant interfaces or structural, non-interface regions. A total of 41% of samples (4705) carried mutations that couldn’t be mapped to any available 3D interaction interface (Figure 2A).\n\nThe analysis revealed wide differences among cancer types in terms of fraction of samples affected by interface mutations. Five cancer types displayed more than 50% of samples carrying significantly impacted Functional Interactions (FIs): UVM (91%), PAAD (75%), SKCM (61%), UCS (57%) and THCA (56%) (Figure 2A). Other cancer types displayed a higher portion of samples affected by mutations at significantly perturbed interfaces which cannot yet be mapped to Reactome processes (e.g. ACC). Finally, cancer types like KIRP, KICH, DLBC and CHOL displayed no samples significantly affected by mutations perturbing any known protein interaction interfaces (Figure 2A).\n\nMore than half of cancer types with significantly affected interaction interfaces (15 out of 28) display only mutations at Cancer Genes Census (CGC) genes22 at FI interfaces (Figure 2B). After loosening the thresholds for enrichment significance, i.e. no False Discovery Rate (FDR) cutoff, we found a total of 3638 (32%) samples carried CGC gene mutations at FI interfaces (Figure 2C). Cancer types with higher proportions of significantly affected FI interfaces also display greater contributions of CGC gene mutations at FI interfaces (e.g. UVM = 92%, UCS = 82%, PAAD = 77% and SKCM=66%). Interestingly, we were able to detect patterns of CGC gene mutations at FI interfaces even in those cancer types with no significantly affected interaction interfaces (e.g. KIRP = 11%, CHOL = 39%, KICH = 17%, DLBC = 43%) (Figure 2C).\n\nWe also assessed whether biological processes, via either individual reactions or whole pathways, were significantly affected by somatic mutations in terms of mediating interaction interfaces. Defining biological processes significantly affected by edgetic mutations,23 instead of individual genes, allowed us to provide a mechanistic interpretation framework for low frequency mutations participating in common biological processes, thus facilitating the understanding of the oncogenetic mechanisms of cancer driver mutations in the long tail distribution.24 A total of 25 cancer types displayed 556 significantly mutated reactions (corresponding to 460 interfaces and 150 genes) and 375 pathways (corresponding to 2397 interfaces and 759 genes) (see Extended data: Tables S2 and S338). A total of 303 reactions displayed at least two genes affected by interface mutations and 20 reactions were found to be affected in at least five genes (Table S2). The reaction “MAP 2Ks and MAPKs bind to the activated RAF complex” in SKCM is the single reaction carrying the largest number of interface mutations (from 179 unique samples; Table S2) affecting participating members (i.e. SRC, MAPK3, NRAS, RAP1A, MAP 2K2, ARAF, BRAF, KSR1, KSR2, RAF1).\n\nWe obtained a landscape of cancer type specific patterns of perturbed processes by mapping each reaction or pathway to its top level biological pathway (Figure 3; Extended data: Figure S138). Signal transduction is the single most affected top level pathway, showing significantly perturbed reactions and pathways in 92% of cancer types (23 out of 25 and 26 out of 28 total cancer types displaying respectively significant reactions and pathways). Signal transduction processes also display high variability in terms of samples mutated and interfaces involved with melanoma (SKCM) involving the highest number of perturbed interfaces when considering either reactions or pathways (Figure 3; Figure S1; firebrick). Given the high number of reactions affected in signaling events, it is easier to visualize significantly affected pathways (Figure 4). As expected, receptor and non-receptor tyrosine kinase pathways are the most widely perturbed signaling pathways. In total, 10 out of 12 pathways from the “Signaling by Receptor Tyrosine Kinases” super-pathway (https://reactome.org/PathwayBrowser/#/R-HSA-9006934&PATH=R-HSA-162582) are affected through cancer-type specific mutation signatures. Signaling by EGFR is the most recurrently perturbed pathway, being affected in 65% (17 out of 26) cancer types. Several other signaling pathways are related to GPCR signaling either through canonical pathways (i.e. GPCR downstream signaling, Gastrin-CREB signaling pathway via PKC and MAPK, GPCR ligand, Signaling by Rho GTPases or PIP3 activates AKT signaling) or through WNT signaling (i.e. Beta-catenin independent WNT signaling, Degradation of beta-catenin by the destruction complex, TCF dependent signaling in response to WNT).\n\nDiameters are proportional to the numbers of unique samples having mutations in top level pathways and color shades are proportional to the numbers of FI interfaces.\n\nDiameters are proportional to the numbers of unique samples and color shades are proportional to the numbers of FI interfaces.\n\nOther top level processes that are widely perturbed are Immune System and Gene Expression (Transcription) (Extended data: Figure S238). Perturbed reactions linking to Gene expression (Transcription) are dominated exclusively by TP53 reactions, which are affected in all the 14 cancer types, with the sole exception of UCS where an additional reaction is affected by PPP2R1A mutations (Figure S2). We found at least one reaction contributing to the Immune System process significantly affected in 84% (21 out of 25) of cancer types, for a total of 19 unique reactions. The majority of significantly perturbed reactions in Immune System (12 out of 19) appear selectively mutated in only one cancer type, suggesting shared steps of dysregulation of a key cancer process (Extended data: Figure S338).\n\nIntriguingly, we also found that a total of 15 significant reactions and 29 significant pathways in 8 and 14 cancer types, respectively, displayed no known CGC cancer drivers among the affected genes and were mutated in 10 or more unique samples in some cases (see Table S2 and S3). For example, we found complement cascade reactions, e.g. “C5b:C6:C7 translocates to the plasma membrane” (Figure 5A), significantly perturbed in 12 HNSCC samples (FDR = 2.8·10−3) through mutations affecting five genes (i.e. C8A, C9, C6, C8B, C8G), which are predicted to disable the interaction with binding partners (Figure 5B). Another reaction of the terminal pathway of complement, “C5b binds C6” (Figure 5A), was found mutated in 11 SKCM samples (FDR = 1.2·10−2) at C5 mediated interfaces, likewise predicted to perturb the interaction with binding partners (Figure 5B). The complement cascade pathway is also overall significantly affected in HNSCC in 15 samples (FDR = 0.01) with no CGC gene mutations reported (Table S3). Additionally, a few other pathways are also affected in multiple cancer types with no mutations in CGC genes, including: Asparagine N-linked glycosylation (STAD FDR = 0.01, KIRC FDR = 0.02), ER to Golgi Anterograde Transport (STAD FDR = 0.02, KIRC FDR = 0.02), GPCR ligand binding (LIHC FDR = 0.003, BLCA FDR = 0.001) and Cilium Assembly (SKCM FDR = 0.03, LUAD FDR = 0.04) (Table S3).\n\nSignificantly affected reactions can be visualized on a reaction network, where nodes represent reactions and edges represent temporal or preceding/following relationships between reactions. Certain network modules are affected in specific cancer types such as STAD (e.g. Figure 6, lower blue module) while others are widely perturbed in multiple tumor contexts. The network view also allows for enhanced visualization of reaction interdependencies. For example, we identified a major cluster suggesting crosstalk between Signal Transduction and Immune System reactions in multiple cancer types (the zoomed region in Figure 6A). The majority of reactions in this cluster are significant in THYM. However, eight of them are significant in multiple cancer types. Reactions within the cluster are largely annotated for signaling transduction but five of them are annotated for immune system. In addition, reactions involving PIK3CA and NRAS (or KRAS), which are recurrently mutated in a mutually exclusive fashion in multiple cancer types, also support a major crosstalk between Signal Transduction and Immune Systems (Extended data: Figures S3 and S438).\n\nReactions in light blue that are not significant are used as linker nodes to link significant reactions together. Cancer types where reactions are significant are shown as bar charts inside reaction nodes with different colors; B) The mapping between the significant reactions and functional interactions of STAD in both the reaction network and the FIN highlighting the advantage of survey protein variants in cancer with multi-scale perspectives.\n\nThe analysis pipeline we developed was used to survey the protein variants in cancer with a multi-scale perspective, allowing us to uncover cancer driver mutations significantly related to protein interactions or biochemical reactions and providing complementary results to help us draw biological insights. A reaction may be expanded into a set of functional interactions while a functional interaction may be involved in multiple reactions. Using STAD as an example (Figure 6B), Reaction 679977 (TP53 family members bind PPP1R13B or TP53BP2, https://reactome.org/PathwayBrowser/#/R-HSA-6799777) can be expanded into four functional interactions (PPP1R13B-TP63, PPP1R13B-TP73, PPP1R13B-TP53 and TP53-TP53BP2), two of which (PPP1R13B-TP53 and TP53-TP53BP2) are statistically significant after FDR correction. Conversely, the interaction between CASP8 and CFLAR is extracted from nine significant reactions that are annotated for programmed cell death (https://reactome.org/PathwayBrowser/#/R-HSA-5357801). Interestingly, this interaction itself is not significant, presumably caused by FDR correction because there were many more protein interactions subject to analysis than reactions. Other interesting examples can be found for STAD in Figure 6B too. For example, Reaction 200421 (activation of cytosolic AMPK by phosphorylation, https://reactome.org/PathwayBrowser/#/R-HSA-200421), which is significant, is mapped to nine FIs that are not significant. The functional interaction between PIK3CA and PIK3R1 is involved in 10 reactions and both the FI and all 10 reactions are significant.\n\nTo explore the translational potential of our approach, we checked whether patients carrying mutations perturbing either individual interfaces, reactions or pathways were found to be statistically associated with patient overall survival. When considering interfaces, we found only one instance and no significant interfaces involving PIK3CA (Extended data: Table S438). Grouping interface perturbing mutations through reactions or pathways increases the statistical power of survival analysis (Extended data: Tables S5 and S638). Indeed, we found six and ten significant pathways in LGG (Brain Lower Grade Glioma) and UCEC (Uterine Corpus Endometrial Carcinoma) (adj P (Cox) < 0.05) respectively, invariably involving PIK3CA along with different combinations of other oncogenes (e.g. NRAS, KRAS) interfaces. Intriguingly, in both LGG and UCEC Signaling by Interleukins pathway has the largest numbers of affected patients (respectively 27 and 105 samples; Table S6). LGG patients with mutations in this pathway exhibit shorter survival (hazard ratio = 2.08, adjusted p-value = 0.05) while UCEC patients show longer survival (hazard ratio = 0.35, adjusted p-value = 0.02), suggesting that patients carrying mutations of genes involved in the same pathways of recurrently mutated oncodrivers might cause different phenotypes (e.g. survival time) in different cancer contexts.\n\nReactomeFIViz19 is Reactome’s Cytoscape app, implementing a suite of features for users to conduct pathway- and network-based data analysis and visualization based on Reactome pathways and its functional interaction network. To assist users to explore and browse the analysis results from this study, we implemented a set of new features in ReactomeFIViz. Figure 7 illustrates three views, allowing users to visualize variants in a multi-scale perspective ranging from pathways and reactions to protein-protein interactions and protein 3D structures. Users can load analysis results in the pathway diagram view to highlight reactions based on adjusted p-values (Figure 7A) and select a specific reaction to inspect functional interactions impacted by mutations and highlighted according to adjusted p-values (Figure 7B). Furthermore, users can also select a specific interaction and visualize mutation locations in the protein 3D structures pulled directly from PDB (Figure 7C). ReactomeFIViz provides results from all TCGA cancers as well as pancancer, from which users can choose one to explore (Figure 7D). For more information on how to use ReactomeFIViz to explore the analysis results, see the user guide at https://reactome.org/userguide/reactome-fiviz#Structural_Variants_Visualization.\n\nA. Pathway diagram with reactions highlighted according to p-values from the structural analysis; B. Functional interactions for the selected reaction in A (in blue) with interactions highlighted according to p-values; C. Protein-protein interaction 3D view with mutation locations displayed in balls; D. Table control for users to choose a specific cancer to visualize its analysis results.\n\n\nDiscussion\n\nIn this study, we conduct a systematic analysis of protein variants in cancer by mapping non-synonymous cancer somatic mutations from TCGA to protein interaction interfaces using Mechismo and the Reactome functional interaction network first, and then mapping those interfaces back into biochemical reactions and pathways to search for statistically significant interactions, reactions and pathways. Mechismo offers the possibility to predict positional effects of mutations and PTMs at biomolecular interfaces without the need to homology model 3D complexes. Reactome is unique in providing a comprehensive set of manually annotated biochemical reactions which can be leveraged to interpret cancer somatic variants in the context of biochemical reactions and biological pathways. Our analysis is focused on biochemical reactions carried out by protein-protein and protein-chemical interactions and it differs from previous published studies, which were focused either on protein-protein interactions or biological pathways, e.g.25 By focusing on biochemical reactions, we identified and provided mechanistic explanations for rare mutations impinging on reactions participating in established onco-pathways involving recurrently mutated oncodrivers (e.g. Receptor Tyrosine Kinase signaling). Our multiscale representation of biomolecular processes was instrumental in highlighting interactions or reactions playing roles in crosstalk between higher level pathways belonging to distinct domains of biological function (e.g. Signal Transduction and Immune Systems). We also identified significantly affected processes not involving any CGC, thereby representing potentially new cancer-associated processes. For example, we identified several interface mutations affecting the complement cascade pathway, which is emerging as a key regulator of the tumor microenvironment fostering tumor growth and metastasis.26 Several of the identified pathways also represent new therapeutic opportunities readily targetable by off-label drugs. For instance, we identified several GPCR signaling pathways characterized by rare mutations on either the receptors or downstream signaling nodes, confirming our previous findings that sparse GPCR mutations might converge in dysregulating signaling cascades similar to recurrent mutations of coupled G-proteins.27,28\n\nOn top of this study, we have implemented a set of new features in ReactomeFIViz for the community to browse and explore the results of our analysis, facilitating the understanding of potential cancer driver mechanisms in a unique, multi-scale perspective that is built upon the Reactome pathway knowledgebase and extends from gene and protein to protein interactions, reactions and pathways. Together with ReactomeFIViz’s previously existing features for visualizing drug-target interactions in the same context of pathways and interaction networks and fuzzy logic simulation to model perturbation of drugs and somatic mutations on pathway activities,29 users are able to conduct in-silico drug screening and modeling in search of potential therapies by targeting significantly impacted reactions and related functional interactions. We will explore the utility of leveraging ReactomeFIViz in this way.\n\nThe study reported in this paper was based on protein-protein and protein-chemical interaction 3D structures. The limited coverage of protein-protein interaction 3D structures still constrains our survey to a small portion of protein-protein interactions extracted from biochemical reactions and pathways annotated in Reactome. In the future, when results and tools from ongoing large-scale protein structure determination30 or prediction projects (e.g. CASP community31) become available, we expect to obtain more insightful results by applying our approach.\n\nIn summary, we have developed a multi-scale approach to investigate protein structure variants in cancer in the context of protein interactions, reactions and pathways annotated in Reactome. This approach is generic and can be applied to study other types of abnormal protein omics features collected from cancer or other complex diseases. For example, abnormal up or down protein or gene expression data or post-translational modifications can be mapped to protein interactions, reactions, and pathways to investigate their potential impact. Our approach can also be easily extended to integrate other types of interactions, such as protein-DNA, protein-RNA and protein-membrane interactions, for which Reactome has annotated many complexes and reactions. We plan to conduct a more complete analysis by including all these interactions as well as integrated mutations and PTMs sets (e.g. from CPTAC proteogenomics studies) in the future.\n\n\nMethods\n\nWe downloaded the latest MAF files of 32 TCGA Cancer types from http://firebrowse.org/ (2016 release), for a total of 1.8M somatic mutations from 7120 patients. A total of 1.79M non-synonymous somatic mutations were mapped on canonical Uniprot sequences, yielding 1.16M unique protein variants. Ony Ensembl transcripts matching in length to corresponding Uniprot sequences were retained using Oncotator32 “UniProt Exact Match” function and only predicted variants whose reference amino acids matched those at corresponding positions in Swissprot sequences were retained for further inspection.\n\nWe adapted the algorithm we developed to build the Reactome FI network21 to extract functional interactions between proteins and proteins or proteins and chemicals from Reactome annotated reactions. The FI network used in this study is different from our previously constructed FI network. Here we limited our analyses to FIs extracted from annotated Reactome reactions only. To construct the Reactome reaction network, we connect two reactions (e.g. reaction1 and reaction2) together if the output from reaction1 is an input, catalyst, or regulator of reaction2. To control spurious connections among reactions, a set of small molecules, including ATP, ADP, Pi, H2O, GTP, GDP, CO2 and H+, is excluded for this reaction connection checking. The version of the Reactome knowledgebase used in this study is release 59, released in December 2016, which may have some annotations that don’t exist in the current Reactome release.\n\nWe mapped protein-protein and protein-chemical functional interactions from Reactome FIN to 3D interaction interfaces from Mechismo (mechismo.russelllab.org).16 We used Uniprot accessions to map protein entities and employed a chemoinformatics similarity search to map chemical entities. In more detail, we retrieved InChI identifiers for both ChEBI33 (cross-referenced to Reactome) and PDB34 (cross-referenced to Mechismo) chemical components. We then generated topological fingerprints for both sets through the RDKit package (https://www.rdkit.org/) using the Tanimoto score as a similarity metric. We considered only interactions with matching Uniprot accessions and with chemical similarity greater than 0.5 as corresponding protein-chemicals interactions.\n\nWe predicted functional consequences of TCGA non-synonymous somatic mutations using Mechismo,16 which matches protein positions to positions within structures and identifies sites affecting interactions with other proteins, DNA/RNA or small-molecules. We considered high confidence predictions for protein-protein interactions including known structures or close (≥70% sequence identity) homologs and representing only very confident, physical protein-protein interactions. For chemical and DNA/RNA we also considered predictions with low/medium confidence (as low as 30% sequence identity).\n\nWe identified the most perturbed interactions in cancer by ranking each interacting pair based on the number of unique samples where a missense mutation was predicted to affect the interface. We tested the significance of the most perturbed interactions in TCGA by using an interactome perturbation random background model (BM). We defined it by first collecting the missense variants found in each sample and randomly shuffling the same substitutions between equivalent positions in the same protein. We obtained equivalent Mechismo data for BM and calculated the probability of obtaining the same number of observed perturbing events for each interaction by chance using a binomial test:\n\nwhere N is the total number of samples, c is the number of unique samples in which a given interaction has been found perturbed and P is the probability of getting the same interface perturbed from the random background distribution. The obtained P-values were adjusted using the FDR/Benjamini-Hochberg procedure.35 We considered instances with adjusted P-values below 0.05 as significant.\n\nWe similarly applied the same BM and statistical test to define reactions and pathways whose mediating FIs' interfaces are significantly affected.\n\nWe downloaded clinical records of TCGA patients using TCGAbiolinks Bioconductor package36 and considered “vital_ status” , “gender”,”days_to_death”, “days_to_last_follow_up”, “age_at_diagnosis”. Cox’s proportional hazard model was employed to predict hazard ratios and survival probability of patients affected by interface-, reaction- and pathway-perturbing mutations, employing age and sex as covariates.\n\nAll the statistical analysis has been done in Python 3.8.11 (www.python.org) through scipy 1.6.2 (www.scipy.org), statsmodels 0.12.2 (statsmodels.sourceforge.net) and lifelines 0.25.9 (lifelines.readthedocs.org/en/latest) libraries.\n\nWe developed a MySQL database to store the analysis results via a Hibernate API (https://hibernate.org) and a RESTful API to serve the analysis results from the MySQL database to ReactomeFIViz using the Spring MVC framework (version 4.3.10, https://spring.io/projects/spring-framework). New user interfaces were added to ReactomeFIViz using Java Swing (Java 8, https://docs.oracle.com/javase/tutorial/uiswing/) by following the standard practice to develop apps for Cytoscape.37 We used Java (JDK 1.8) for software programming and host the source code at GitHub: https://github.com/reactome-fi/mechismows for the RESTful API and https://github.com/reactome-fi/CytoscapePlugIn for the front-end user interfaces in ReactomeFIViz.\n\n\nData availability\n\nMAF files of the 32 TCGA cancer types available from http://firebrowse.org/ (2016 release)\n\nThe outcome of the analysis is available through the Reactome FIViz Cytoscape app: https://reactome.org/tools/reactome-fiviz\n\nThe mysql database dump used for the server-side application: https://doi.org/10.5281/zenodo.5590953.\n\nZenodo: Leveraging biochemical reactions to unravel functional impacts of cancer somatic variants affecting protein interaction interfaces, https://doi.org/10.5281/zenodo.5598224.38\n\nThis project contains the following extended data:\n\n• Figure S1: Significantly affected pathways in individual TCGA cancer types mapped to top level Reactome pathways. Diameters are proportional to the numbers of unique samples having mutations in top level pathways and color shades are proportional to the numbers of FI interfaces.\n\n• Figure S2: Significantly affected reactions belonging to the Gene expression top level pathway. Diameters are proportional to the numbers of unique samples and colors are proportional to the numbers of FI interfaces.\n\n• Figure S3: Significantly affected reactions belonging to the Immune System top level pathway. Diameters are proportional to the numbers of unique samples and colors are proportional to the numbers of FI interfaces.\n\n• Figure S4: Significantly affected reactions belonging to the Signal Transduction top level pathway. Diameters are proportional to the numbers of unique samples and colors are proportional to the numbers of FI interfaces.\n\n• Table S1: Mutation enrichment at protein interaction interfaces.\n\n• Table S2: Mutation enrichment at Reactome Reactions interfaces.\n\n• Table S3: Mutation enrichment at Reactome Pathway interfaces.\n\n• Table S4: Survival analysis of significantly affected interactions.\n\n• Table S5: Survival analysis of significantly affected reactions.\n\n• Table S6: Survival analysis of significantly affected pathways.\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).\n\n\nSoftware availability\n\nHome page for user guide describing procedures on how to use ReactomeFIViz features for visualizing cancer somatic mutations via a multi-scale perspective: https://reactome.org/userguide/reactome-fiviz#Structural_Variants_Visualization\n\nCytoscape app store: http://apps.cytoscape.org/apps/reactomefiplugin\n\nLatest source code: https://github.com/reactome-fi/CytoscapePlugIn (ReactomeFIViz app code) and https://github.com/reactome-fi/mechismows (Server-side code)\n\nSource code as at the time of publication: https://github.com/reactome-fi/CytoscapePlugIn/releases/tag/f1000_cancer_2021 and https://github.com/reactome-fi/mechismows/releases/tag/F1000_Cancer_2021\n\nArchived source code as at the time of publication: https://doi.org/10.5281/zenodo.5590945 39 (ReactomeFIViz app code) and https://doi.org/10.5281/zenodo.5590949 40 (Server-side code)\n\nLicense: Creative Commons Attribution 4.0 International (CC BY 4.0) License (https://reactome.org/license).",
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}
|
[
{
"id": "135297",
"date": "10 May 2022",
"name": "Andras Szilagyi",
"expertise": [
"Reviewer Expertise Bioinformatics",
"protein structure",
"protein-protein interactions",
"pathway analysis"
],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nIn this impressive work, the authors investigated all available data on somatic cancer mutations by connecting 3D protein-protein and protein-chemical interface data with data on biochemical reactions and pathways, thereby revealing significantly impacted reactions and pathways. This novel approach has yielded numerous important insights. Analysis of patient survival times identified mutations perturbing processes affecting patient survival. The authors also created an updated visualization tool enabling the users to explore the results of the analyses.\nThe methodology is sound, and the results support the conclusions. There are only a few technical problems which need to be fixed:\nThe supplementary figures (Extended data) cannot be evaluated because they got cropped, probably during some conversion step.\n\nThe top part of Fig. 5 is not labeled, and the legend does not refer to it.\n\nOn Page 3, 'ERBB2' is misspelled as 'ERRB2'.\n\nThe resolution of Fig. 6 is poor and most labels are unreadable. Since this figure is very complex, a vector image (preferably SVG) would be better. The legend says some nodes contain bar charts, but in fact they are pie charts. Actually, bar charts would be better because there are too many similar colors in the pie charts.\n\nThe supplementary tables are provided in an Excel file which is inconvenient to use as the column labels are not fully visible. Please increase the height of the row for the column labels and use wrapped text.\n\nThe cancer types are indicated with acronyms, but they are not expanded anywhere. Please provide their expansions, or a link where these can be found.\nRegarding the survival analysis, the authors used the clinical records from TCGA. Were there any missing data? If yes, how were they handled?\nThis article is a very significant contribution. However, it cannot be fully evaluated until the supplementary figures are fixed.\n\nIs the work 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": "9060",
"date": "30 Nov 2022",
"name": "Guanming Wu",
"role": "Author Response",
"response": "Dear Dr. Szilagyi, Thanks a lot for reviewing our manuscript. We have made the following updates according to your comments: The supplementary figures (Extended data) cannot be evaluated because they got cropped, probably during some conversion step. Our mistake. This has been fixed now. The top part of Fig. 5 is not labeled, and the legend does not refer to it. The top part of Fig 5 is deleted. On Page 3, 'ERBB2' is misspelled as 'ERRB2'. Thanks! Fixed. The resolution of Fig. 6 is poor and most labels are unreadable. Since this figure is very complex, a vector image (preferably SVG) would be better. The legend says some nodes contain bar charts, but in fact they are pie charts. Actually, bar charts would be better because there are too many similar colors in the pie charts. Thanks for pointing this out. We replaced the figure with a PDF version, which is scalable. Furthermore, we provided the original Cytoscape file (Figure_6_Networks.cys) in Extended Data for readers to investigate the networks using Cytoscape, which can be downloaded from https://cytoscape.org/. We tried to use bar charts in Cytoscape. However, we believe using pie charts is better. Also we fixed the name of the charts to “pie charts” from “bar charts”. Thanks a lot for your suggestion! The supplementary tables are provided in an Excel file which is inconvenient to use as the column labels are not fully visible. Please increase the height of the row for the column labels and use wrapped text. Thanks for the suggestions. We have increased the height of the row for the column labels and use wrapped text. The cancer types are indicated with acronyms, but they are not expanded anywhere. Please provide their expansions, or a link where these can be found. Regarding the cancer types acronyms, these are the standard abbreviations provided by TCGA (https://gdc.cancer.gov/resources-tcga-users/tcga-code-tables/tcga-study-abbreviations). We provided a reference in the “TCGA dataset” section of the Methods. Regarding the survival analysis, the authors used the clinical records from TCGA. Were there any missing data? If yes, how were they handled? Thanks for pointing this out. We didn’t consider in our analysis samples with missing values. We have now made this point clear in the “Survival analysis” section of the Methods. Thanks again for your comments."
}
]
},
{
"id": "147340",
"date": "20 Sep 2022",
"name": "Pier Luigi Martelli",
"expertise": [
"Reviewer Expertise Computational biology",
"Bioinformatics",
"Structural and functional characterization of proteins and their variants",
"Machine and deep learning."
],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe paper by Raimondi et al. presents an analysis aiming at elucidating the effect of somatic, cancer-related variants on interaction interfaces of complexes involved in biochemical reactions. Data from 7120 patients of 32 cancer types are downloaded from TGCA and analysed with Mechismo for determining the possible functional effect of variations at protein-protein interfaces. Functional interfaces are then mapped on Reactome reactions and pathway and an enrichment procedure is adopted to highlight the most relevant biochemical processes involved in each cancer type.\nThe paper is generally clearly written. In my opinion, paper readability would benefit from a more extended review of the methods at the basis of Mechismo and of the definition of functional interfaces. In particular, it is unclear what is the expected number of variations that Mechismo can annotate on proteins without 3D structure, as reported in the introduction. For the 1.16 M unique variations from TCGA, authors only report the number of those mapped on known 3D structures, and it is unclear whether they include those deriving from homology-based analysis. If yes, it would be useful to provide some more detail and discuss possible differences of effect predictions depending on the similarity between the wildtype sequence and the adopted structure.\nOther minor points: - Introduction: the sentence \"The gene-level analysis tells us what changes occur in genes or proteins, but not the functional impact\" is a bit misleading, since different approaches have been developed for predicting possible impact at different levels (function, stability...)\n- Legends of figures could be improved, adding details. In particular it is not clear what \"donor\" means in Figure 2.\n- Supplementary figures S1-S4 are unreadable in the current version.\n\nIs the work 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": "9061",
"date": "30 Nov 2022",
"name": "Guanming Wu",
"role": "Author Response",
"response": "Dear Dr. Pier Luigi Martelli, Thanks a lot for reviewing our manuscript. We have made some changes according to your comments. Please see our reply to your comments below: In my opinion, paper readability would benefit from a more extended review of the methods at the basis of Mechismo and of the definition of functional interfaces. In particular, it is unclear what is the expected number of variations that Mechismo can annotate on proteins without 3D structure, as reported in the introduction. For the 1.16 M unique variations from TCGA, authors only report the number of those mapped on known 3D structures, and it is unclear whether they include those deriving from homology-based analysis. If yes, it would be useful to provide some more detail and discuss possible differences of effect predictions depending on the similarity between the wildtype sequence and the adopted structure. We mapped mutations to 3D structures, either known or homologous, by considering UniProt alignment to PDB fasta sequences via BLAST, considering matches with E-value threshold of 0.0001, and storing the best match for each position in the UniProt sequence. Then we considered high confidence predictions for protein-protein interactions including known structures or close (>=70% sequence identity) homologs and representing only very confident, physical protein-protein interactions. For chemical and DNA/RNA we also considered predictions with low/medium confidence (as low as 30% sequence identity). We have made these points clearer both in the Results and Methods sections. Introduction: the sentence \"The gene-level analysis tells us what changes occur in genes or proteins, but not the functional impact\" is a bit misleading, since different approaches have been developed for predicting possible impact at different levels (function, stability...) When we said “gene-level analysis”, we meant the analysis is about genes or proteins such as somatic mutations or structural variants. Indeed, many approaches have developed to predict impacts of mutations on protein stability and functions. But we’d like to argue that “stability” is still a protein-level feature. To know the impact of mutation on the protein’s function, extra information is needed (e.g. what pathways or biological processes have been annotated for the protein, etc.) for higher level analysis (e.g. pathway enrichment analysis). Regardless, we added some phrases (as shown below in parentheses) to the original sentence. Now this sentence reads as the following: The gene-level analysis tells us what changes (e.g. structural variants) occur in genes or proteins, but not the functional impact (e.g. biological processes)… Legends of figures could be improved, adding details. In particular it is not clear what \"donor\" means in Figure 2. Thanks for pointing this out. We have changed “donors” to “patients” in Figure 2. Supplementary figures S1-S4 are unreadable in the current version. These figures have been fixed. It is our mistake. Thanks again for reviewing our manuscript and comments."
}
]
}
] | 1
|
https://f1000research.com/articles/10-1111
|
https://f1000research.com/articles/11-1459/v1
|
09 Dec 22
|
{
"type": "Research Article",
"title": "Significant demotivating factors affecting Saudi EFL students: An investigative study with PYP students",
"authors": [
"Fahd Hamad Alqasham"
],
"abstract": "Background: Motivation has long been seen as a deciding factor in the learning of a foreign language and in ensuring success for the learners. As a corollary to this, demotivation adversely affects learning success and negatively influences learning outcomes. The present study examines the factors that negatively affect Saudi EFL learners.\n\nMethods: The study subjects are 45 male and 35 female learners, and the data were collected using a 50-item questionnaire. The factors under study are course instructor, course-content and facilities, attitude towards EFL and socio-cultural factors. Data have been analyzed using the SPSS software.\n\nResults: Results show that the factors around socio-cultural, and course instructor (M=4.06, 3.67) respectively were the most influential in aggravating the student's demotivation. Furthermore, experience of failure is at play as a moderate factor (M=2.84) whereas the course content and facilities and attitude towards EFL factors, (M=2.57; M=1.83) are perceived as less influential factors in demotivating the EFL students. Findings also show a marginal non-significant difference between male and female participants regarding perceptions of the demotivating factors (males = 5.30, females= 5.70); the difference is not significant, P=, .834.\n\nConclusions: It is presumed that the findings of this study might be helpful for EFL learners and instructors of Saudi Arabia in devising coping strategies to overcome demotivation.",
"keywords": [
"Demotivation",
"EFL Learners",
"Foreign Language",
"gender",
"Motivation",
"PYP students"
],
"content": "Introduction\n\nMastering English as a foreign language is an overt goal for students in Saudi Arabia. However, the actual proficiency level in English is far from satisfactory in this country irrespective of which school or university is under scrutiny. As per the findings of Education First English Proficiency Index (EF EFI, 2019), the status of Saudi Arabia was 98th out of 100 non-native English-speaking countries in 2019 but improved to 83rd last year, showing an upward movement of 15 positions. Many scholars have investigated the reasons behind the poor performance of EFL learners in Saudi Arabia. They found that the teaching and learning process seems to be imperfect and unsatisfactory owing to various factors (Khan, 2011). They have the opinion that foreign language learning has the direct impact of many positive and negative factors. Among the numerous positive factors is motivation which has been at the center of investigation for a long time.\n\nMotivation researchers focused on the role of motivation in learners’ interest enhancement in learning (Al-Hoorie & Macintyre, 2020; Dörnyei, 2020; Reeve, 2013). However, a new concept has taken birth in recent times: the idea of demotivation in ESL/EFL acquisition which is taken as another aspect of motivation (Falout & Maruyama, 2004; Kikuchi & Sakai, 2009). It is important to note that de-motivation has been understood, defined, investigated, and viewed in different ways. Dörnyei (2001) conceptualized these learning-detrimental factors as ‘demotivating factors’ and they are responsible for derailing the progress of EFL-learners. De-motivation, according to Trang and Baldauf (2007) is now gaining attention in the field of foreign or second language learning. Emphasizing the need for understanding demotivation, Albalawi (2017) remarks that the concept holds great significance for L2 teachers and learners. The present study examined the factors of demotivation prevalent among Saudi EFL learners. The conventional view about de-motivation is that it is signified by low motivation or a third type of motivation rather than a phenomenon in its own rights (Trang & Baldauf, 2007). It is seen as arising out of lack of adequate motivation to accomplish a specific goal (Alahdal & Al Ahdal, 2019; Sakai & Kikuchi, 2009; Soureshjani & Riahipour, 2012).\n\nLack of zest and zeal, enthusiasm, willingness, excitement, fervor are the symptoms of de-motivation as identified by researchers. It is the lack of effort, need and desire directed towards the learning process (Aydin, 2012). It is the process of the accumulation of negative factors encaging students gradually and steadily. During the process of second language learning, students’ motivation may relate to many negative influences, about which Dörnyei and Ushioda (2011) stated, ‘learning related particular events or experiences, such as performance, anxiety, public humiliation, heavy work demands or poor test results and factors in the social learning environments, such as the personality and the attitude of the teacher or classroom counter-cultures and peer pressures.’ Furthermore, Dörnyei (2005) states in this regard that demotivation ultimately hinders the learning, as well as ignoring the positive learning factors.\n\nDemotivated learners are those who were motivated in the past but owing to some negative factors they lose interest in learning. Reasons behind the loss of interest is called de-motives that are the negative counterpart of motives. But it is important to note that all negative factors cannot be labeled as de-motives. Dörnyei (2001) has mentioned three negative factors that are not taken as de-motives as they do not carry negative value. These are factors of distraction instead of reducers for motivation. For example, watching movies instead of doing home-work is not the result of demotivation. Secondly, steady loss of interest is not taken as de-motive because de-motives are the specific factors that reduce motivation. Thirdly, there are some compelling situations that prove detrimental in goal accomplishment. If a person does not join a course, simply because it is time-consuming or very costly then this will not be termed as a de-motive. So, such deliberate action cannot be termed as demotivation. Researchers admit demotivation is ‘another side of motivation’ (Dörnyei & Ushioda, 2011; Sakai & Kikuchi, 2009).\n\nThere is a plethora of de-motives that hinder the EFL learners’ language acquisition, and these are also the root cause of demotivation. Pointing out the various factors that marginalized the performance of Saudi EFL learners, Khan (2011) mentions mother tongue interference, inadequate target language exposure, late language exposure, poor learning environments, poor learner autonomy, learners’ lifestyle, issues of discipline, punctuality, motivation, needs assessment, social pressure and status, lack of guidance and excessive freedom. Studies related to demotivating factors have brought out many demotivating factors for EFL learners in the length and breadth of the world but research into demotivation factors in the Arab world, in particular, in the Saudi Arabian context seems to be scarce (Alyousif & Al-Suhaibani, 2021).\n\nFrom the above discussion on demotivation, it is clear that knowledge of demotivating factors may prove to be a boon for EFL learners. It is worthy to note that most of the studies related to demotivation talk about university students (for example, Alyousif & Alsuhaibani, 2021). The present study is related to the students who have just graduated from high school and are enrolled in the preparatory year program (PYP) section of the department of English at university level. It is expected to be of great use to the EFL learners who will join the undergraduate courses in different universities of the Kingdom of Saudi Arabia. The participants chosen in this study are those students who could not pass the exam or dropped out due to their failure to pass the exam. The present study aims to realistically highlight the strongest demotivating factors that may help teachers and institutions in constructing healthy frameworks for motivating learners and promoting learning.\n\n\nLiterature review\n\nSignificance, essentiality and vitality of motivation to achieve required and desired success in EFL learning has been unequivocally admitted by many researchers. Motivation is an essential factor in foreign language learning and in learners’ target-language proficiency achievement its role is crucial (Ghadirzadeh et al., 2013; Hu, 2011; Zhang, 2007). Likewise, Broussard and Garrison (2004) considered motivation as an attribute that presupposes action or inaction of the learner. Pointing towards the value of motivation, Littlewood (1987) affirmed that “motivation is the critical force which determines whether a learner embarks on a task at all, how much energy he devotes to it and how long he preserves” (p. 53). A good amount of research exists on different aspects of motivation with varied conclusions highlighting both internal and external factors and these, in turn, can start, sustain, intensify, or discourage behaviors (Reeve & Deci, 1996). However, as a consequence of the recent studies in L2 acquisition – a new concept known as demotivation has come to the fore which is taken as another side of motivation (Dörnyei & Ushioda, 2011; Falout et al., 2009; Sakai & Kikuchi, 2009). However, despite of its importance, demotivation needs greater attention from researchers (Dörnyei, 2001).\n\nDemotivation has been defined in the Cambridge Dictionary as the “lack of interest in and enthusiasm about your work” (Cambridge University Press, 2020). Falout and Falout (2005) believed that if motivation pushes learning for life, demotivation cuts learning short. Furthermore, Kaivanpanah and Ghasemi (2011) stated that “any failure to learn a second language may be largely due to the existence of demotivating factors on the part of learners” (p. 90). It proves to be a stumbling block in EFL students’ learning success. A great many teachers find learners demotivated in classrooms in various educational contexts (Ghadirzadeh, et al., 2012, p.189). Demotivation has scarcely been investigated in various contexts (Dörnyei, 2001; Falout, 2012; Falout & Maruyama, 2004; Kikuchi & Sakai, 2009; Tsuchiya, 2006). Dörnyei (2005) admitted the absence of research on demotivation, ‘… in spite of their great significance, demotivation has received very little attention either in mainstream psychology or L2 research.’\n\nAmong the important research studies conducted on the topic of demotivation are the studies of Dörnyei (1998), Zhang (2007), Kikuchi and Sakai (2009), Gorham and Millete (1997), Kim (2011), Arai (2004), Falout et al. (2009) and Tsuchiya (2006). Kikuchi and Sakai (2009) identified five demotivating factors for Japanese EFL learners namely feeble intrinsic motivation, lackluster school facilities, learning materials and contents, test scores and teachers’ methodology. Content, learning material and test score were at the top of the demotivating factors contrary to the teachers’ competency and teaching styles which were the main demotivating factors found in several earlier studies. Findings from Zhang (2007) conducted in America, Germany, China and Japan concluded that teachers’ inadequate skill and incompetence are the main demotivating factors. Moreover, shortcoming in teachers’ behavior was found as the main demotivating factor in the research work of Kim (2011) and others. Other important research on demotivation among EFL learners have been conducted by (Arai, 2004; Dörnyei, 1998; Falout et al., 2009; Tsuchiya, 2006; Ikeno, 2002) who found that the negative attitude towards foreign language, low level of self-confidence, teaching techniques, teachers’ competence, personalities, inadequate school facilities, interference of another foreign language, attitude of group members and the compulsory nature of learning the foreign language are the important demotivating factors.\n\nBekleyen (2011) studied the demotivating factors for Turkish university EFL learners and concluded that the absence of latest teaching technology, course-books, unskilled teachers, and teaching techniques are the main demotivating factors. Furthermore, Kim and Kim (2015) conducted exploratory research for Korean EFL learners and in addition to a few other motivational factors, they found three crucial demotivating factors, namely: negative perceptions towards affordance, difficulties in English learning and pressures of exams. Similar research was carried out by Meshkat and Hassani (2012) to identify the demotivating factors prevalent among Iranian high school EFL learners wherein the study concluded that teaching strategies, unskilled teachers, lack of facilities, dearth of intrinsic motivation, learning context and material and test scores were the visible demotivating factors.\n\nIn addition to the above-mentioned studies that were conducted in countries other than the Arab world, here is a birds’ eye view of the significant studies that have dealt with the demotivating factors affecting Arabic EFL learners’ language learning endeavors (Al-Khasawneh, 2017; Al-Asmari & Javid, 2011; Dhaif-Allah, 2005; Qashoa, 2006; Javid, 2010; Mohammad H. Al-khairy, 2013; Alyousif & Alsuhaibani, 2021). Qashoa (2006) carried out a study to investigate the demotivating and motivating factors for the EFL learners of UAE secondary school students. Findings of this research stated that demotivating factors for the secondary school EFL learners are related to teachers’ characteristics, teaching methods, course books, peer pressure, low level of self-confidence, social and religious points of view along with vocabulary, grammar and spelling.\n\nMost recently, Alyousif & Alsuhaibani (2021) investigated the demotivating factors that affected Saudi high school EFL students. The findings showed that subject related and teacher related demotivating factors as the prominent demotivating factors for Saudi EFL learners. In another study of the same nature that was conducted by Al-Khairy (2013) wherein the demotivating factors affecting the Saudi undergraduates were investigated, it was found that ignorance related to the importance of English, negative attitude towards English, the use of Arabic in EFL class, course content, and lack of opportunities for speaking English were the significant demotivating factor.\n\nOn the basis of researchers’ findings on demotivation among EFL learners, it is crystal clear that it emerges from both intrinsic and extrinsic factors. Many researchers such as (Dörnyei, 2001, 2005; Jomairi, 2011) have considered the extrinsic factors while some other researchers (Ushioda, 2011; Kim & Seo, 2012) have emphasized the intrinsic factors. Significant findings regarding intrinsic demotivating factors are lack of self-confidence and the negative attitude of learners themselves towards EFL learning (Ikeno, 2002). Regarding the extrinsic demotivating factors, Dörnyei (2005) identified them as external factors that have an adverse effect on learner motivation and their desire to learn or engage in the learning process.\n\nTsuchiya’s study (2006) highlighted nine demotivating factors comprising both intrinsic and extrinsic factors that compel negative attitudes towards the English-speaking community, negative attitude towards English itself, teachers, classes, negative group attitude, the compulsory nature of English study, reduced self-confidence, ways of learning and lack of English-speaking models. Likely, Falout and Maruyama (2004) studied the causes of demotivation in terms of high and low proficiency students and found that low proficiency learners don’t regard extrinsic factors as demotivating as the high proficiency students.\n\nFrom the above discussion it is clear that most of the studies are silent on the impact of some prominent variables such as gender, culture, economic, social and age factors. The present study aims to examine the causes of demotivation arising out of the gender, social, cultural and environmental issues along with those common issues related to teachers, students, course content and facilities and experience of failure. It is a well- known fact that Saudi Arabia has a specific social-cultural background (Al-Khairy, 2013) and here the EFL learners could not accomplish and display tangible results as expected (Al-Khairy, 2013; Al-Jarf, 2008). So, in the present study, special attention has been paid to investigate whether the social and cultural factors play a role in aggravating demotivation among PYP EFL learners of Saudi Arabia to fill the research gap. It answers these two questions:\n\n\nResearch questions\n\n\n\n1. What are the chief factors responsible for demotivation amongst the EFL/L2 learners in Saudi Arabia?\n\n2. Do the demotivational factors have the same effect on the Saudi EFL male and female learners?\n\n\nMethods\n\nThe present research was conducted with the help of ex-post facto design. It was conducted in Saudi Arabia, Methnab, Qassim region during the Academic year (1443 AH; May 2022). This research design was necessitated as the manipulation and selection of the independent variables were beyond the control of the researcher.\n\nThe researcher contacted all the potential participants who could not pass the Intensive Course Program and sent them the questionnaire. These participants were from the registration office data including 120 leavers, however a final sample of 45 males and 35 females participated and responded to the questionnaire. It is to be noted that soon after enrolment, they either dropped or failed in the Intensive Course Program (ICP) of the department of Translation & English in College of Sciences and Arts, Methnab, Qassim University, KSA. Prior verbal consent was obtained for participation. The researcher obtained verbal consent with the help and approval of the Scientific and Ethical Committee in the Department. All participants were adult Saudi nationals, and the age average of the group was 20 years.\n\nAnonymity of information was assured, and concealment of identity was guaranteed to the participants in order to maintain research ethics. The researcher obtained ethical approval from the Scientific and Ethical Approval in the Department of English Language and Translation, Methnab College of Sciences and Arts, Qassim University. The consent letter was submitted to the Head of the English Department (May 23, 2022) who subsequently followed up the procedures including scheduling a time for the researcher to meet with the respondents to explain to them the aim of the study; the respondents thus expressed their consent to the publication of their responses.\n\nTo identify the main demotivating factors found among the EFL learners of KSA, a 50-item questionnaire related to demotivation was prepared by the researcher. A copy of the questionnaire can be found under Extended data (Alqasham, 2022). The questionnaire was designed using Google Drive Forms. The link to the questionnaire was shared with the participants. Two weeks were given to them to complete the questionnaire. They were informed that after they complete the questionnaire to press submit at the end of the form. This was modeled as per the questionnaire developed by (Falout & Maruyama, 2004; Sakai & Kikuchi, 2009). The questionnaire was recorded on a 5-point Likert scale with values ranging from 1-5 (1= strongly disagree to 5= strongly agree). The items loaded onto five de-motive categories: (1) attitude toward EFL learning, Item 1-5, (2) experience of failure, items 6-19, (3) course content and facilities, item 20-29, items 30-42, (4) course instructor, and (5) social-ecological factors, items 43-50. The reliability coefficients for the demotivation questionnaire are recorded in Table 1 and were found acceptable at (0.601 into 0.784) with an average of (0.648).\n\nBasic statistical tools were applied to test the hypothesis of the present study. Descriptive statistical data were obtained (Mean, Standard deviation and Rank) and compared through statistical software of SPSS, version 26 that was performed for data analysis. Varimax rotation procedure was used as it enables the researcher to examine the frequency and order of importance of demotivating factors affecting the participants’ progress in EFL classes of Saudi Arabia. An independent sample t-test was obtained to measure the detect whether there is a difference in demotivation factor attributed to sex.\n\n\nResults\n\n1. RQ1: What are the chief factors responsible for demotivation amongst the EFL/L2 learners in Saudi Arabia?\n\nTable 2 presents the descriptive data of the students' attitudes towards the five demotivational factors. Table 2 shows that the chief factors are social-cultural and course instructor. Students perceived them to a high extent. They scored for socio-cultural factor M=4.06, Std= 1.18, and for course instructor M=3.67, Std=1.27. Furthermore, students’ perceived experience of failure as a moderate factor scored M=2.84, Std=1.47. The course content and facilities (M=2.57, Std=1.32), and attitude towards EFL (M=1.83, Std=1.05) were perceived as low factors that had an effect on motivation. The full dataset can be found under Underlying data (Alqasham, 2022).\n\n2. RQ2: Do the demotivational factors have the same effect on the Saudi EFL male and female learners?\n\nTable 3 displays the denotational factors effects for male and female learners, parsed gender-wise. An independent sample t-test was performed to analyze the effects of demotivational factors on gender. Table 3 reveals that females scored in all the factors M=5.70, Std=1.168 whereas males’ perceptions reached M=5.70, Std=1.168. This showed that the demotivational factors have very few higher effects on female learners, but the effect is not significant statistically. The t-test was used to calculate the difference. This reached .834, P>.05.\n\n\nDiscussion\n\nThe investigation showed that two demotivational factors impacted learners to a great degree. They are social-cultural, and course instructor (M=4.06, 3.67) respectively. Regarding the course instructor factor, lack of skill and competency in course instructors were found to be a major demotivating factor for the EFL learners in many studies. The present study also found many issues related to the teachers are the causes of demotivation. So, this study confirms the findings of much recent research (e.g., Alqasham, 2022; Arai, 2004; Zhang, 2007; Bekleyen, 2011; Kim and Seo, 2012; Evans & Tragant, 2020; Falout & Maruyama, 2004; Dörnyei & Ushioda, 2011). They reported that the social and cultural factors also have a big influence in aggravating demotivation. Furthermore, the socio-cultural factor is also found amongst the chief factors that inhibit EFL students' learning. Ushioda (1994) conducted a study at Trinity College of Dublin in Ireland to investigate the demotivating factors for EFL learners and found that the demotivating experiences were predominated by the external factors associated with the learning environment. This finding is in line with the findings of the present study which found socio-cultural factors to be a great demotivating factor. Saudi society is highly traditional, and it is deeply rooted in the Arabic language. In social gatherings, parks, banks, commercial centers, airports, festivals, banquets, marriages, business, and trade, only Arabic is used. On the contrary, in the department of English, with some rare exceptions, the language of communication is English. Most of the parents do not recognize the importance of English while the students do not use English at all. In Saudi schools, students and teachers do not communicate in English. Teaching of the language is solely for the purpose of examination. English is detached from society so when the students join ICP after completing high school and are asked to speak, listen, read, and write in English (which they have never done in the past), they are likely to fail. In most colleges, seventy to eighty percent of students fail in the intensive English learning course program.\n\nThe study found that demotivational factors have a very slight effect on female learners over male learners. However, the effect of such factors was not statistically significant. It was greater than (.005). The higher effect on female learners compared to male learners may be interpreted according to their psyche as well as to the unique experience and upbringing of females in Arabic culture. Bin-Hady and Al-Tamimi (2021) reported that due to the Yemeni heritage as a conservative society where female students are not allowed to talk with male students on social media and in some family, female students were not allowed to own a mobile phone. They are more respected and cared for in the Saudi context, therefore, the same demotivation factors affected female students more than male students. However, as the probability vale (Sig.= .834) is not significant, we cannot be certain in our claim. This finding contradicts Tavanapour and Chalak's (2019) who studied the impact of the motivational factors on sex in the Irani context for both students and teachers. The study showed that both students and teachers were demotivated alike. Furthermore, the study indicated that male learners were more affected by the demotivational factors than female learners.\n\n\nConclusions\n\nFrom the above discussion it is clear that demotivating factors do not arise only out of the factors related to teachers, course content, passive attitude towards EFL and experience of failure, but also from the socio-cultural structure of the highly traditional Saudi society. There is a need to extend the use of English from the classrooms to society. Demotivation arises from the fact that English is not used anywhere as a language of communication in the society. It is left to the mercy of classroom teachers who are doing more harm than good by teaching in a hackneyed way. There is a need to improve both on intrinsic and extrinsic fronts. A healthy environment wherein students, parents and society realize the value of English needs to be created. To change the classroom situation, it is essential to recruit skilled and dynamic teachers. Saudi society is highly civilized, and its students do not point out the instructors’ negligence out of respect. There is a need to make the teachers realize the value of new teaching techniques. One important demotivating factor arises from the nature of the Saudi classroom. Generally, in the EFL classes in Saudi Arabia students of various proficiency levels are enrolled, but teachers do not know how to handle the mixed ability classes and, as a consequence, only highly proficient students study. So, there should be some special training for the course instructors by reputed scholars so that they can teach effectively in a class of mixed ability. Class size also should be decreased for better individual attention. Teachers are unable to redress the problems of all the students in a large class, consequently most of the students, especially the low proficiency students, fail to follow the lectures. Also, there is need to revise the course content. The topics of study should have native elements so that students may enjoy it. Proper attention should be paid on intrinsic and extrinsic factors to divorce demotivation and live with motivation. There is need to bring changes in the environment for the betterment of EFL learners. There should be extra-curricular activities in which English language and its students should be encouraged to participate. Group study should be encouraged in the classroom. Home assignment should be given on a regular basis. There should be monitoring of the EFL classes by the learned teachers and supervision by the higher authorities with appreciation, encouragement, and needful directions.\n\nThis research was conducted in the Department of English & Translation of the College of Arts & Science (Qassim University of KSA) and a total of 80 EFL learners (45 male and 35 female) from one undergraduate program participated. This hardly depicts a comprehensive view of the different demotivating factors faced by the EFL learners enrolled in different educational institutions in KSA. It is also important to note that this research is based on the descriptive statistics of the quantitative data only which cannot unveil the whole face of demotivation deeply rooted in numerous factors. Limited variables have been studied. Other variables such as age, different academic specialization, and students’ level of proficiency should be considered to obtain more comprehensive results.\n\nFuture research on the issue of demotivation should be conducted in various institutions applying different research methods so that more demotivating factors may come to the fore. Further, varied factors that affect motivation should be studied and teachers made aware of these. Class size should be reasonable and mixed abilities classes should be avoided as far as possible. Social and cultural background of the Saudi-EFL learners should be studied to find the real causes of general demotivation for EFL learning.",
"appendix": "Data availability\n\nFigshare: Significant demotivating factors affecting Saudi EFL Students: An Investigative Study with PYP students. https://doi.org/10.6084/m9.figshare.20732359.v2 (Alqasham, 2022).\n\nThis project contains the following underlying data:\n\n- Demotivates - Underlying Data.xlsx [These data include denotative factors on five axes. The data was loaded onto five de-motive categories: (1) attitude toward EFL learning, Item 1-5, (2) experience of failure, items 6-19, (3) course content and facilities, item 20-29, items 30-42, (4) course instructor, and (5) social-ecological factors, items 43-50]\n\nFigshare: Significant demotivating factors affecting Saudi EFL Students: An Investigative Study with PYP students. https://doi.org/10.6084/m9.figshare.20732359.v2 (Alqasham, 2022).\n\nThis project contains the following extended data:\n\n- Questionnaire\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).\n\n\nReferences\n\nAlahdal A, Al-Ahdal AAMH: Effectiveness of collaborative learning as a strategy in the teaching of EFL. Opción: Revista de Ciencias Humanas y Sociales. 2019; 35(20): 1026–1043.\n\nAl-Asmari AA, Javid CZ: Motivational constructs: A cross sectional study of EFL students at Taif University. J. Stud. Soc. Sci. Humanit. 2011; X1X(2): 73–194.\n\nAl-Khairy MH: English as a foreign language learning de-motivational factors as perceived by Saudi undergraduates. Eur. Sci. J. 2013; 9(32): 365–382.\n\nAlbalawi F: L2 demotivation among Saudi Learners of English: The role of the language learning mindsets. University of Nottingham;2017. (Unpublished PhD dissertation).\n\nAl-Hoorie AH, MacIntyre PD: Language motivation theory. Multilingual Matters;2020.\n\nAl-Jarf R: The Impact of English as an International Language (EIL) upon Arabic in Saudi Arabia. Asian EFL J. 2008; 10(4)Reference Source\n\nAl-Khasawneh FM: Demotivating factors Affecting EFL Learning of Saudi Undergraduate Students. International Journal of Language Education and Applied Linguistics. 2017; 6(5): 25–34. Publisher Full Text\n\nAlqasham FH:Significant demotivating factors affecting Saudi EFL Students: An Investigative Study with PYP students. figshare. Dataset. 2022. Publisher Full Text\n\nAlqasham FH: Investigating English as a foreign language learners’ perceptions, emotions, and performance during online collaborative writing. Front. Psychol. 2022; 13: 954011. Publisher Full Text\n\nAlyousif R, Alsuhaibani Z: English Language Learning Demotivating Factors for Saudi High School EFL Students. Engl. Lang. Teach. 2021; 14(8): 29–39. Publisher Full Text\n\nArai K: What ‘demotivates’ language learners? Qualitative study on de-motivational factors and learners’ reactions. Bulletin of Toyo Gakuen. 2004; 12(3): 39–47.\n\nAydin S: Factors causing demotivation in EFL teaching process: A case study. Qual. Rep. 2012; 17: 1–13. Article 101.\n\nBekleyen N: Demotivating factors in the EFL environment. Frontiers of Language and Teaching. 2011; 2: 151–156.\n\nBin-Hady WRA, Al-Tamimi NOM: The use of technology in informal English language learning: evidence from Yemeni undergraduate students. Learning and Teaching in Higher Education: Gulf Perspectives. 2021; 17(2): 107–120. Publisher Full Text\n\nBroussard SC, Garrison MB: The relationship between classroom motivation and academic achievement in elementary-school-aged children. Fam. Consum. Sci. Res. J. 2004; 33(2): 106–120. Publisher Full Text\n\nDhaif-Allah A: An exploration of Saudi students' integrative and instrumental motivation for learning English. Occasional Papers in the Development of English Language Education. 2005; 39: 55–113.\n\nCambridge University Press: Domestic. Cambridge English Dictionary;2020.Reference Source\n\nDörnyei Z:Demotivation in foreign language learning. Proceedings of the TESOL’98 Congress. Seattle, WA:1998.\n\nDörnyei Z: Motivational strategies in the language classroom. UK:Cambridge University Press;2001.\n\nDörnyei Z: The psychology of the language learner. Mahwah, NJ:Lawrence Erlbaum;2005.\n\nDörnyei Z: Innovations and challenges in language learning motivation. Routledge;2020.\n\nDörnyei Z, Ushioda E: Teaching and researching: Motivation. Routledge;2011.\n\nEvans M, Tragant E: Demotivation and dropout in adult EFL learners. TESL –EJ. 2020; 23(4).\n\nFalout J, Elwood J, Hood M: Demotivation: Affective states and learning outcomes. System. 2009; 37(3): 403–417.\n\nFalout J, Falout M:The other side of motivation: Learner demotivation. JALT 2004 conference proceedings. Tokyo:2005; (pp. 280–289).\n\nFalout J, Maruyama M: A comparative study of proficiency and learner demotivation. Lang. Teach. 2004; 28: 3–9.\n\nFalout J: Coping with demotivation: EFL learners’ re-motivation processes. TESL-EJ. 2012; 16(3): 1–29.\n\nGhadirzadeh R, Hashtroudi FP, Shokri O: Demotivating factors for English language learning among university students. J. Soc. Sci. 2012; 8(2): 189–195.\n\nGhadirzadeh R, Hashtroudi FP, Shokri O: Study of the effective factors on the university students’ underachievement in English language learning. Engl. Lang. Teach. 2013; 6(11): 122–129. Publisher Full Text\n\nGorham J, Millete DM: A comparative analysis of teacher and student perceptions of sources of motivation and demotivation in college classes. Commun. Educ. 1997; 46(4): 245–261. Publisher Full Text\n\nHu R: The relationship between demotivation and EFL learners’ English language proficiency. Engl. Lang. Teach. 2011; 4(4): 88–96.\n\nIkeno O: Motivating and demotivating factors in foreign language learning: A preliminary investigation. Ehime University Journal of English Education Research. 2002; 2: 1–19.\n\nJavid CZ: Addressing the causes that hinder effective English Language teaching in Saudi Universities: A case study. Bani Swaif University Journal. 2010; 80: 479–513.\n\nJomairi S: Demotivating factors in second language learning at State, Azad and Payam-Nour Universities. International Conference on Languages, Literature and Linguistics IPEDR. 2011; (Vol. 26: pp. 300–303).\n\nKaivanpanah S, Ghashemi Z: An investigation into sources of demotivation in second language learning. Iran. J. Appl. Linguist. 2011; 14(2): 89–110.\n\nKhan I: Learning difficulties in English: Diagnosis and pedagogy in Saudi Arabia. Educ. Res. 2011; 2(7): 1248–1257.\n\nKikuchi K, Sakai H: Japanese Learners’ demotivation to study English: A survey study. JALT Journal. 2009; 31(2): 183–204.\n\nKim S: Testing a revised measure of public service motivation: Reflective versus formative specification. J. Public Adm. Res. Theory. 2011; 21(3): 521–546.\n\nKim TY, Kim YK: Elderly Korean learners' participation in English learning through lifelong education: Focusing on motivation and demotivation. Educ. Gerontol. 2015; 41(2): 120–135. Publisher Full Text\n\nKim TY, Seo H: Elementary school students’ foreign language learning demotivation: A mixed methods study of Korean EFL context. Asia-Pac. Educ. Res. 2012; 21(1): 160–171.\n\nLittlewood W: Foreign and second language learning: Language acquisition research and its implications for the classroom. Cambridge University Press;1987.\n\nMeshkat M, Hassani M: Demotivating factors in learning English: The case of Iran. Procedia. Soc. Behav. Sci. 2012; 31: 745–749. Publisher Full Text\n\nQashoa SHH: Motivation among learners of English in the secondary schools in the eastern coast of the UAE. The British University in Dubai;2006. (Unpublished PhD. dissertation).\n\nReeve J:Extrinsic rewards and inner motivation. Handbook of classroom management. Routledge;2013; (pp. 655–674).\n\nReeve J, Deci EL: Elements of the competitive situation that affect intrinsic motivation. Pers. Soc. Psychol. Bull. 1996; 22(1): 24–33.\n\nSakai H, Kikuchi K: An analysis of de-motivators the EFL, classroom. System. 2009; 37(1): 57–69. Publisher Full Text\n\nSong Y: Motivation and demotivation in L2 learning. Sino-US English Teaching. 2005; 2(7): 79–81.\n\nSoureshjani KH, Riahipour P: Demotivating factors on English speaking skill: A study of EFL language learners and teachers’ attitudes. World Appl. Sci. J. 2012; 17(3): 327–339.\n\nTanaka M: Examining EFL vocabulary learning motivation in a demotivating learning environment. System. 2017; 65: 130–138. Publisher Full Text\n\nTavanapour S, Chalak A: Relationship between Demotivation Factors and Gender among Iranian EFL Students and Teachers in Iranian Academic Contexts. J. New Adv. Engl. Lang. Teach. Appl. Linguist. 2019; 1(2): 116–135.\n\nTrang TTT, Baldauf RB Jr: Demotivation: Understanding resistance to English language learning-the case of Vietnamese students. J. Asia TEFL. 2007; 4(1): 79–105.\n\nTsuchiya M: Factors in demotivation of lower proficiency English learners’ at College. The Kyushu Academic Society of English Education (KASELE). 2006; 34(1): 87–96.\n\nUshioda E: Language learning motivation, self and identity: Current theoretical perspectives. Comput. Assist. Lang. Learn. 2011; 24(3): 199–210. Publisher Full Text\n\nUshioda E: L2 motivation as a qualitative construct. Teanga. 1994; 14: 76–84.\n\nZhang Q: Teacher misbehaviors as learning de-motivators in College Classrooms. A cross-cultural investigation in China, Germany, Japan, and United States. Commun. Educ. 2007; 56(2): 209–227. Publisher Full Text"
}
|
[
{
"id": "157885",
"date": "21 Dec 2022",
"name": "Wagdi Rashad Ali Bin-Hady",
"expertise": [
"Reviewer Expertise TEFL",
"TESOL",
"ELT",
"Applied linguistics",
"ICT"
],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 the manuscript is well written. It investigated one of the most fundamental in English language learning. Some points for the development of the paper:\nIn the abstract, the author is recommended to mention the research design.\n\nThere are spaces within the in-text citations, e.g., ( Aydin, 2012). This needs to be omitted.\n\nThe first paragraph of the literature review, which is devoted to defining motivation, is not related to the research; I think it is likely to be omitted.\n\nIn the results section, there is an error in writing the mean scores of males (Table 3 reveals that females scored in all the factors M=5.70, Std=1.168, whereas males’ perceptions reached M=5.70, Std=1.168). Male scored 5.30 not 5.70.\n\nIs the work 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": "203455",
"date": "13 Dec 2023",
"name": "Reza Anggriyashati Adara",
"expertise": [
"Reviewer Expertise applied linguistics",
"demotivation",
"motivation"
],
"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 allowing me to review this manuscript. The title reflects the study very well. In addition, the manuscript provides clear descriptions of the study conducted, allowing other researchers to conduct it in their own contexts. This manuscript is also appropriately designed for demotivation research.\n\nNevertheless, I personally have some reservations regarding the manuscript.\nFirst, the manuscript uses less contemporary sources. Some sources even dated back twenty years ago.\n\nSecond, despite being sufficiently designed to study demotivation in the context of EFL learners, the manuscript does not explain how the researchers interpret the mean score results. It will be difficult for other researchers to use the current manuscript as an example for their future studies.\n\nThird, there is a considerable repetition of ideas in the introduction and literature review. The writers keep bringing demotivation factors, showing a lack of organization.\n\nFourth, the introduction does not show the novelty of the study, making the urgency of the study vague.\n\nFifth, the second research question seems less appropriate. Examining the effects of demotivation is more appropriate for experimental studies.\n\nSixth, the writers do not elaborate the type of sampling used in this study. The writers should explain it in the method section.\n\nSeventh, the discussion section is not explored well, showing a lack of analysis. Besides that, the conclusion section does not provide sufficient implications of the study.\n\nLastly, the manuscript needs an editor to polish the language.\n\nI hope the above comments do not discourage the writers from editing this manuscript as they have potentials to contribute more in the field of English as a second/foreign language, especially in the context of Saudi Arabian learners.\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? No",
"responses": []
},
{
"id": "203419",
"date": "18 Apr 2024",
"name": "Sahib Khatoon Thaheem",
"expertise": [
"Reviewer Expertise English Language Teaching"
],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nAlthough this paper contains many good parts, the beginning of the abstract needs amendment. The abstract must explain the methods and sample. Analysis with digits is not to be presented in the abstract; only major findings should be shown here. Recommendations and implications should be included in the abstract.\nThe literature review is mostly 10 or 12 years back; it should be a recent 5 years.\nLiterature review of 2021, 2022, 2023 is not included.\nAnalysis is well explained but there should be recommendations and implications properly explained. In the discussion, synthesis should be well-knitted.\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": []
}
] | 1
|
https://f1000research.com/articles/11-1459
|
https://f1000research.com/articles/11-130/v1
|
01 Feb 22
|
{
"type": "Clinical Practice Article",
"title": "The cocktail infection: anaerobic and aerobic co-Infections in tubercular vertebral osteomyelitis",
"authors": [
"Shyamasunder N Bhat",
"Raghuraj Kundangar",
"Nishanth Ampar",
"Anika Sait",
"Padmaja Ananth Shenoy",
"Cynthia Amrutha Sukumar",
"Kavitha Saravu",
"Shyamasunder N Bhat",
"Raghuraj Kundangar",
"Nishanth Ampar",
"Anika Sait",
"Padmaja Ananth Shenoy",
"Kavitha Saravu"
],
"abstract": "Background: Anaerobic organisms have been known to have an association with dental infections, bacteremia, endocarditis and soft tissue infections. However, anaerobic isolation from bone and joint infections are relatively rare. Sparse literature has been found on reports of anaerobic osteomyelitis. There is no literature reported on anaerobic osteomyelitis complicating Tuberculosis of spine. Case Report: We report two cases of tuberculosis of spine complicated by aerobic and anerobic infections. The first is a case of a young female who presented with chronic lower backache and fever. Examination revealed a lumbar scoliosis with paraspinal tenderness. Magnetic resonance imaging (MRI) of the spine showed lumbar spondylodiscitis with multiple abscesses. There were air-fluid levels noted in the abscesses. The pus sent for CBNAAT (cartridge based nucleic acid amplification test) was positive. Further the cultures also grew Escherichia coli (aerobic) Bacteroids fragilis and Peptoniphilus asachrolyticus (anaerobic) organisms. She improved with a course of intravenous antibiotics and decompression surgery. The second case is a middle aged man who presented with chronic neck pain and fever. Examination revealed kyphosis of the neck with spasm of the neck muscles and midline tenderness. MRI showed C4-5 cervical spondylodiscitis with parapharyngeal collections showing air-fluid levels. The pus culture showed Streptococcus constellatus (aerobic) and Prevotella sps. (anaerobic). The CBNAAT report was positive for Mycobacterium tuberculosis. The patient was treated with intravenous antibiotics and cervical decompression. Conclusion: Though tubercular vertebral osteomyelitis (TVO) is usually a diagnosis in itself, it should not hinder us from considering secondary infections (both aerobic and anaerobic) complicating the osteomyelitis. Further, the presence of air-fluid levels on imaging studies and the presence of foul smell during operative exploration of the spine must arouse the suspicion of an anaerobic co-infection. Isolation and treatment of these organisms are crucial as they may hamper the clinical outcome of the primary TVO.",
"keywords": [
"Anaerobic osteomyelitis",
"Prevotella",
"Bacteroides",
"Peptostreptococcus",
"spondylodiscitis",
"tuberculosis of Spine",
"Tubercular Vertebral Osteomyelitis"
],
"content": "Introduction\n\nAnaerobic organisms have been known to have an association with dental infections, bacteremia, endocarditis and soft tissue infections.1 However, anaerobic isolation from bone and joint infections are relatively rare. This could be attributed to the fastidious nature of the organism which makes isolation from the bone and joint infections cumbersome.1\n\nThe most common organisms implicated in anaerobic osteomyelitis are Bacteroides, Fusobacterium, Peptostreptococcus and Clostridium species. Predisposing factors include children, contiguous spread of infection, vascular disease and complicated fractures.2 Sparse literature has been found on reports of anaerobic osteomyelitis. There is no literature reported on anaerobic osteomyelitis complicating Tuberculosis of spine/Tubercular vertebral osteomyelitis (Pott’s spine). However, our cases highlight the rare isolation of gram-negative anaerobes (Prevotella, Bacteroides, Peptoniphilus) from Tubercular vertebral osteomyelitis (TVO) in immunocompetent adults diagnosed with tuberculosis of spine.\n\n\nCase report\n\nA 22-year-old poorly built (body mass index: 16 kg/m2) lady from rural South India who was pursuing her undergraduate degree, presented with complaints of low back ache of one-month duration. It was insidious in onset and gradually radiated to the right gluteal region. She was able to walk only a few steps with a limp. There was no bowel or bladder disturbance. She preferred to keep her right hip flexed to about 30 degrees in supine position. The backache was associated with high grade fever with chills and rigors. There was no history of tuberculosis in her family or her community. She had no significant history suggestive of immunosuppression. She had no known co-morbidities. There was no significant genetic history given by the patient.\n\nOn examination, she was febrile (102° F) with a pulse rate of 98 beats/minute. There was midline and right paraspinal tenderness from L3 to sacral region. Neurological examination of lower limbs was unremarkable.\n\nLab investigations showed that she was anaemic (Haemoglobin of 8 g/dl). Her total counts were 13600/mm3 (predominantly neutrophilia). The ESR was elevated (88 mm/hour) and CRP (261mg/L). The renal function tests were within normal limits, while serum albumin was only 1.9 mg/dl. Blood and urine cultures were sterile.\n\nA radiograph revealed mild lumbar scoliosis to the left side and bulky psoas muscle in the right side (Figure 1a and b).\n\nMRI spine revealed features suggestive of spondylodiscitis of L5-S1, spondylitis of L4 with abscesses adjacent to L4, L5, S1, S2 and S3 vertebra in the right side. There was also air-fluid level in the abscess (Figure 1c and d).\n\nShe underwent decompression of L5 vertebra and exploration of L5-S1 space with drainage of the abscess. Intra-operatively foul-smelling gas escaped from the abscess. The L5-S1 disc also had purulent collection. Pus was sent for CBNAAT (cartridge based nucleic acid amplification test) for tuberculosis, aerobic and anaerobic cultures. Material from L5-S1 disc was sent for histopathology.\n\nOn the third hospital day CBNAAT report was suggestive of Mycobacterium tuberculosis she was started on anti-tubercular therapy (ATT). The next day the aerobic culture grew Escherichia coli whereas anaerobic culture grew Bacteroids fragilis and Peptoniphilus asachrolyticus. In addition, she was also started on injection Piperazillin-Tazobactem and oral tinidazole for 10 days. There was wound dehiscence on tenth postoperative day.\n\nThe repeat culture from the pus drained grew only E. coli with similar sensitivity though the growth was scanty. We continued antitubercular drugs and added Injection Amikacin and Imipenem (as per the culture sensitivity report) for a total of three weeks. She was taken up for secondary suturing during this period. Patient was discharged subsequently. On follow-up at one month, her symptoms had improved and her anemia had improved to 9.5 gm%. She had also gained 1.6 kgs in weight. Repeat radiographs and MRI were done at the three-month follow-up and showed resolution of the abscesses. Patient was adherent and tolerated the anti-tubercular medications well.\n\nA 54-year-old man from an urban district of South India, a tailor by occupation, came with complaints of insidious onset of neck pain of about three weeks duration. The pain was gradually progressive over the past 10 days. The pain would aggravate on movements of the neck, but there was no radiculopathy. There was also associated fever with chills and rigors. He reported significant weight loss in the recent past. There was no history of medical co-morbidites. There was no relevant psycho-social or genetic history obtained from the patient.\n\nOn examination, neck appeared short and kyphotic. There was midline tenderness from C4-C7 region. Movements of the neck was grossly restricted due to pain and spasm. The elbow flexion was MRC Grade 4/5 on both sides. There was no sensory deficits and reflexes were normal. He was anaemic (Hb 10 g/dl) with neutrophilia. ESR was moderately elevated at 68 mm/hour.\n\nIn an X-ray of the cervical spine, we noted increased prevertebral soft tissue shadow and erosion of vertebral end plates at C4-5 level with gross kyphosis. Air-fluid levels were also seen in the prevertebral soft tissue shadow (Figure 2a and b).\n\nMRI of the cervical spine confirmed spondylodiscitis of C4-C5 with cord compression. Multiple collections in parapharyngeal space communicating with the pre-vertebral space were also seen along with air-fluid levels (Figure 2c and d)\n\nBy this time the blood culture grew Streptococcus constellatus and he was started on Injection ceftriaxone (2gm q24h) as per sensitivity report. His urine culture was sterile and Brucella agglutination test was negative.\n\nHe underwent anterior cervical decompression, by C5 corpectomy and fusion (Figure 2e and f). A distinct foul-smelling odour was noted intra-operatively and around 100 ml of pus was drained. The C4 and C5 vertebrae were completely destroyed with surrounding caseous material. Pus was sent for aerobic and anaerobic cultures and the caseating tissue was sent for histopathology. Patient tolerated the procedure well.\n\nThe aerobic bacterial culture from the pus grew Streptococcus constellatus with similar sensitivity as that of his blood culture. Anaerobic culture grew Prevotella sps. The CBNAAT report was positive for Mycobacterium tuberculosis. Histopathology confirmed granulation tissue with epitheloid cells and Langhans giant cells suggestive of tubercular osteomyelitis.\n\nHe was started on Antitubercular therapy and Injection Metronidazole (500 mg q8h for two weeks) in addition to Injection Ceftriaxone (two weeks). The wound healed well. Antitubercular therapy is being continued and patient is doing well at two months follow up. Improvement in anemia and ESR as well as weight gain was noted in this patient.\n\nConsent\n\nWritten informed consent for publication of their clinical details and/or clinical images was obtained from the patients.\n\n\nDiscussion\n\nAnaerobic osteomyelitis was first reported in 1884 by Von Langenbeck in case of vertebral osteomyelitis.3 Decades later, Taylor and Davies noted the presence of anaerobic organisms within sequestra in 55% of patients with chronic osteomyelitis.4 It was found that these anaerobic organisms were more frequently isolated from the inside of the sequestra and were usually mixed isolates i.e two or more anaerobic isolates were isolated. The chronicity of the osteomyelitis was directly proportional to the frequency of anerobic isolates found.\n\nIn a retrospective study done on osteomyelitis by Lewis et al., it was found that 39 percent of patients with osteomyelitis had anaerobic infections.1 This serves to prove that anaerobes play a much larger role in osteomyelitis than known previously. It is likely that these infections are less reported due to poor awareness of their prevalence and the cumbersome methods of isolation.\n\nHowever, these organisms were found as a part of the mixed flora which included gram positive cocci, gram negative bacilli and other anaerobes. This is consistent with the pattern of mixed infections reported in our case report. Our first patient was found to have anaerobic Prevotella infection from epidural abscess with associated Streptococcus bacteremia in a chronic tubercular vertebral osteomyelitis. Our second case was also found to have a combination of anaerobes comprising of Bacteroides, Peptoniphilus (Peptotreptococcus) and Clostridium with gram-negative Pseudomonas aeruginosa isolated from a psoas abscess secondary to a chronic tubercular vertebral osteomyelitis.\n\nThe predisposing factors contributing to anaerobic osteomyelitis usually include children, diabetes mellitus, oral infections or procedures, upper respiratory tract infections, trauma, peripheral neuropathy and complicated fractures.2 It is interesting to note that both our patients had no pre morbidities to predispose them to anaerobic osteomyelitis.\n\nIn a review by Raff and Melo of a large series of 193 anaerobic osteomyeltits cases collected from the world literature published between 1936 to 1976, it was the found that the most common anaerobe implicated in osteomyelitis was Bacteroides followed by Peptostreptococcus and Fusobacterium.5 This trend was confirmed by a study by Ziment et al. who found the most common isolate being Bacteroides followed by Peptostreptococcus and Fusobacterium.6 This similar trend of occurrence was noted in a large study done on 134 cases of pyogenic osteomyelitis by Haider et al. between 1992 to 1993.7 This trend agrees with our case of anaerobic osteomyelitis which showed a mixed anaerobic isolate of Bacteroides with Peptostreptococcus and Clostridium.\n\nA review of literature on available data on anaerobic osteomyelitis with Prevotella isolates shows only nine cases reported till date.8–16 Among this only one case reports secondary Prevotella infection on chronic tubercular vertebral osteomyelitis. A summary of the data available on Anaerobic osteomyelitis with prevotella isolates are shown in Table 1.\n\n* Species could not be identified.\n\nBoth our cases did not show any evidence of hematogenous osteomyelitis. This finding concurs with evidence found by in a review study on 61 cases of anaerobic osteomyelitis of long bones17 where 29.5% of the cases were hematogenous and 32.8% were exogenous osteomyelitis. The study by Lewis et al. also shows a preponderance towards exogenous osteomyelitis.1\n\nManagement of anaerobic osteomyelitis includes a two-pronged approach with adequate drainage of purulent material and parenteral administration of antibiotics for at least four to eight weeks.18 The antibiotics were decided according to the sensitivity pattern noted on culture.\n\nStrengths and limitations: The strength of this case series is the similar clinical presentation of both the cases and the presence of aerobic and anaerobic infections in both of them. However, we were unable to follow up the second case beyond two months as he opted for care under a local orthopaedician due to travel constraints. This was one of the limitations of our case series. As a result of this adherence and tolerance of the treatment could not be assessed in the second case.\n\n\nConclusion\n\nThough tubercular vertebral osteomyelitis is usually a diagnosis in itself, it should not hinder us from considering secondary infections (both aerobic and anaerobic) complicating the osteomyelitis. Further, the presence of air-fluid levels on imaging studies and the presence of foul smell during operative exploration of the spine must arouse the suspicion of an anaerobic co-infection. Isolation and treatment of these organisms are crucial as they may hamper the clinical outcome of the primary TVO. Therefore, the cocktail of organisms, both aerobic and anaerobic isolated in these cases highlights the importance of suspecting, isolating and treating these lurking organisms in cases of tuberculosis of spine.\n\n\n\n1. I was suffering from severe back ache for over 2 years with recurrent fever. I was unable to continue my studies also as sitting in class was very painful. I received treatment at several local hospitals but the fever persisted and the pain was not alleviated. Following admission at Kasturba hospital, Manipal, I underwent an MRI of my spine and the doctors found the source of infection in my spine. I underwent a decompression surgery with treatment with TB medicines and intravenous medications at the hospital. The fever subsided in the first week following treatment. Gradually I was able to move around with support and at my 3 month follow-up, I walked without pain to the hospital. I am much better now and have rejoined college to continue my studies.\n\n2. I had neck stiffness and pain for many years. The pain radiated to my arms and I struggled to sit and work for long hours with my neck bent forward. In the hospital, a team of doctors examined me and suspected a spine infection. An MRI confirmed infection of the neck bones and pus collections. I underwent a surgery on my neck and received a long course of medicines in the hospital and at home. I noted a significant improvement in my pain and fever. I was symptom free by my 2-month follow-up at the hospital.",
"appendix": "References\n\nLewis RP, Sutter VL, Finegold SM: Bone infections involving anaerobic bacteria. Medicine. 1978; 57(4): 279–306. PubMed Abstract | Publisher Full Text\n\nBrook I, Frazier EH: Anaerobic osteomyelitis and arthritis in a military hospital: A 10 year experience. Am. J. Med. 1993; 94(1): 21–28. PubMed Abstract | Publisher Full Text\n\nBick E: The source book of orthopaedics. Baltimore: The Williams & wilkins co; 1948.\n\nTaylor K, Davies M: Persistence of bacteria within sequestra. Ann. Surg. 1917; 66: 522–528. PubMed Abstract | Publisher Full Text | Free Full Text\n\nRaff MJ, Melo JC: Anaerobic osteomyelitis. Medicine. 1978; 57: 83–103. Publisher Full Text\n\nZiment I, Miller LG, Finegold SM: Nonsporulating anaerobic bacteria in osteomyelitis. Antimicrob. Agents Chemother. 1967; 7: 77–85. PubMed Abstract\n\nHaider AM, Sundus SB, Thamer AH: Anaerobic osteomyelitis. East. Mediterr. Health J. 1996; 2: 494–500. Publisher Full Text\n\nSurbled M, Perrier-Creach C, Rabouille Y, et al.: Spondylodiscitis caused by Bacteroides melaninogenicus. Presse Med. 1992; 21: 1870–1871.\n\nSalavert M, Navarro V, Roig P, et al.: Vertebral osteomyelitis by Prevotella melaninogenica, Candida albicans, and Mycobacterium tuberculosis in a intravenous drug addict. Enferm. Infecc. Microbiol. Clin. 1997; 15: 117–119.\n\nFukuoka M, Aita K, Aoki Y, et al.: Pyogenic vertebral osteomyelitis caused by Prevotella intermedia. J. Infect. Chemother. 2002; 8: 182–184. PubMed Abstract | Publisher Full Text\n\nSchöber W, Horger M, Niehues D: One case of Gram-negative anaerobic spondylodiscitis with Prevotella intermedia. Arch. Orthop. Trauma Surg. 2003; 123: 436–438. PubMed Abstract | Publisher Full Text\n\nMukhopadhyay S, Rose F, Frechette V: Vertebral osteomyelitis caused by Prevotella (Bacteroides) melaninogenicus. South. Med. J. 2005; 98: 226–228. PubMed Abstract | Publisher Full Text\n\nSalliot C, Lavie F, Azria A, et al.: Retroperitoneal fibrosis secondary to spondylodiscitis after infection with Prevotella. J. Rheumatol. 2005; 32: 957–958. PubMed Abstract\n\nPurushothaman B, Lakshmanan P, Gatehouse S, et al.: Spondylodiscitis due to Prevotella associated with ovarian mass - a rare case report and review of literature. World Neurosurg. 2010; 73: 119–122. PubMed Abstract | Publisher Full Text\n\nHuang CR, Lu CH, Chuang YC, et al.: Clinical characteristics and therapeutic outcome of Gram-negative bacterial spinal epidural abscess in adults. J. Clin. Neurosci. 2011; 18: 213–217. Publisher Full Text\n\nHemant G, Shitij A, Sneha M, et al.: Vertebral osteomyelitis and epidural abscesses caused by Prevotella oralis: a case report. Braz. J. Infect. Dis. 2012 Dec; 16(6): 594–596. Publisher Full Text\n\nTempleton WC, et al.: Anaerobic osteomyelitis of long bones. Rev. Infect. Dis. 1983; 5(4): 692–712. PubMed Abstract | Publisher Full Text\n\nBrook I: Microbiology and management of joint and bone infections due to anaerobic bacteria. Journal of Orthopaedic Science: Official Journal of the Japanese Orthopaedic Association. 2008; 13(2): 160–169. PubMed Abstract | Publisher Full Text"
}
|
[
{
"id": "121806",
"date": "10 Mar 2022",
"name": "Kaouther Maatallah",
"expertise": [
"Reviewer Expertise Rheumatology"
],
"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\nThese are two cases of tuberculosis of the spine complicated by aerobic and anaerobic infections. The first is lumbar spondylodiscitis in a young woman. A second case is a middle-aged man with C4-5 cervical spondylodiscitis. Both cases improved after receiving antibiotic treatment and decompression surgery.\nIn both cases, the chest radiography findings were not detailed. In the first case, no MRI images showed spondylodiscitis.\nThe reason for the surgical treatment is not justifiable since, in both cases, there was no motor deficit.\n\nIs the background of the cases’ history and progression described in sufficient detail? Yes\n\nAre enough details provided of any physical examination and diagnostic tests, treatment given and outcomes? Partly\n\nIs sufficient discussion included of the importance of the findings and their relevance to future understanding of disease processes, diagnosis or treatment? Partly\n\nIs the conclusion balanced and justified on the basis of the findings? Partly",
"responses": [
{
"c_id": "8940",
"date": "04 Jan 2023",
"name": "Cynthia Sukumar",
"role": "Author Response",
"response": "NOT APPROVED These are two cases of tuberculosis of the spine complicated by aerobic and anaerobic infections. The first is lumbar spondylodiscitis in a young woman. A second case is a middle-aged man with C4-5 cervical spondylodiscitis. Both cases improved after receiving antibiotic treatment and decompression surgery. In both cases, the chest radiography findings were not detailed. In the first case, no MRI images showed spondylodiscitis.- MRI images included The reason for the surgical treatment is not justifiable since, in both cases, there was no motor deficit. Is the background of the cases’ history and progression described in sufficient detail? Yes Are enough details provided of any physical examination and diagnostic tests, treatment given and outcomes? Partly - Details included Is sufficient discussion included of the importance of the findings and their relevance to future understanding of disease processes, diagnosis or treatment? Partly - Discussion has been revised as per your suggestions Is the conclusion balanced and justified on the basis of the findings? Partly - Revised"
}
]
},
{
"id": "123606",
"date": "22 Mar 2022",
"name": "Gazanfar Rahmathulla",
"expertise": [
"Reviewer Expertise Traumatic brain and spine injury",
"spinal surgery and associated pathology",
"brain tumors",
"radiosurgery"
],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe authors present a case report on 2 cases of vetebral osteomyelitis of the lumbar and cervical spine where the patients have co-existing mycobacterial and aerobic/anerobic infections. Both cases had surgical intervention with short term reported good outcomes.\nPros - interesting article discussing the presence of overlapping bacterial infections in vertebral osteomyelitis.\nCons - short follow up, no discussion about the length of anti-TB medications used, absence of radiological follow-up.\nThe authors can write their article in standard format of introduction, material / methods where they can note the consent requirement rather than before the discussion.\nThe authors should expand their discussion to explain the duration of anti-TB treatment in the presence of treatment for other organisms, their follow-up strategy and explain how the patient could have got these poly organisms to infect the spine.\nIn the MRI images for case 1, the descriptions can be more clear and detailed, as they only show the psoas abscess but would better serve the readers by focusing on the vertebral osteomyelitis components as well.\nThe authors should place another table with literature discussing TB with other infections and all the papers available on this topic. There will not be many and hence this paper would then be strengthened.\nGrammatical changes required in different paragraphs.\n\nIs the background of the cases’ history and progression described in sufficient detail? Yes\n\nAre enough details provided of any physical examination and diagnostic tests, treatment given and outcomes? No\n\nIs sufficient discussion included of the importance of the findings and their relevance to future understanding of disease processes, diagnosis or treatment? Yes\n\nIs the conclusion balanced and justified on the basis of the findings? Yes",
"responses": [
{
"c_id": "8938",
"date": "04 Jan 2023",
"name": "Cynthia Sukumar",
"role": "Author Response",
"response": "The case report has been revised to include physical examination, diagnostic tests, details of treatment and clinical outcome. Additional radiological images have been included for case 1 to indicate spondylodiscitis. Literature search to find similar cases with tubercular vertebral osteomyelitis and anaerobic infection was also done and details included in the manuscript."
},
{
"c_id": "8939",
"date": "04 Jan 2023",
"name": "Cynthia Sukumar",
"role": "Author Response",
"response": "APPROVED WITH RESERVATIONS The authors can write their article in standard format of introduction, material / methods where they can note the consent requirement rather than before the discussion.- revised The authors should expand their discussion to explain the duration of anti-TB treatment in the presence of treatment for other organisms, their follow-up strategy and explain how the patient could have got these poly organisms to infect the spine.- Revised In the MRI images for case 1, the descriptions can be more clear and detailed, as they only show the psoas abscess but would better serve the readers by focusing on the vertebral osteomyelitis components as well. - images included The authors should place another table with literature discussing TB with other infections and all the papers available on this topic. There will not be many and hence this paper would then be strengthened. Grammatical changes required in different paragraphs. Is the background of the cases’ history and progression described in sufficient detail? Yes Are enough details provided of any physical examination and diagnostic tests, treatment given and outcomes? No... Details provided in the revised manuscript Is sufficient discussion included of the importance of the findings and their relevance to future understanding of disease processes, diagnosis or treatment? Yes Is the conclusion balanced and justified on the basis of the findings? Yes"
}
]
}
] | 1
|
https://f1000research.com/articles/11-130
|
https://f1000research.com/articles/11-1098/v1
|
27 Sep 22
|
{
"type": "Research Article",
"title": "Modelling time-varying volatility using GARCH models: evidence from the Indian stock market",
"authors": [
"Farman Ali",
"Pradeep Suri",
"Tarunpreet Kaur",
"Deepa Bisht",
"Pradeep Suri",
"Tarunpreet Kaur",
"Deepa Bisht"
],
"abstract": "Background: In this study, we examined the volatility of the Indian stock market from 2008 to 2021. Owing to the financial crisis, volatility forecasting of the Indian stock market has become crucial for economic and financial analysts. An empirical study of the returns of the NSE indices revealed an autoregressive conditional heteroskedastic trend in the Indian stock market. Methods: Using GARCH 1, 1 (generalized autoregressive conditional heteroskedasticity) and FIGARCH (fractionally integrated GARCH), we examine investor behaviour and the persistence of long-term volatility. Results: The empirical findings of the estimated models suggest that shocks persist for a long time in NSE returns. Furthermore, bad news has a greater impact on stock volatility than good news. The return on assets is stable but highly volatile, even though the Indian economy has experienced the global crash to some extent. Conclusions: Models of volatility derived from the GARCH equation provide accurate forecasts and are useful for portfolio allocation, performance measurement, and option valuation.",
"keywords": [
"GARCH Model",
"Stock market",
"Volatility",
"NSE Return",
"Financial Crisis"
],
"content": "1. Introduction\n\nIn recent years, both academics and financial analysts have shown an increasing interest in modelling and forecasting the volatility of financial time series, which is an increasingly fertile area of behavioural finance research. Since volatility affects many economic and financial applications, such as portfolio optimization, risk and returns analysis and asset pricing, it is a highly relevant concept. Asymmetry in the volatility process of unanticipated shocks is a prominent feature of financial market time series. Negative news often has a greater impact on the conditional variance of equity returns than positive news due to the leverage effect (Black, 1976). Globalizing the interdependence and size of major financial markets, the transmission of financial market information to India has become a subject of interest. An economic spillover occurs when one event sets off another event in a similar way, impacting economies both within and outside the country (Nandy & Chattopadhyay, 2019). In 2008, when Lehman Brothers collapsed in the US, a domino effect was created and hit economies worldwide, including India. Currently financial markets are closely interconnected and driven by trust (Prasad & Reddy, 2009). In times of crisis, investors make errors of judgment as long as a group of investors make irrational decisions, leading to the worsening of the situation in the stock market. Developed and emerging markets have been a riveting field of research on behavioural finance owing to interlinked stock markets worldwide. Coronavirus disease (COVID-19) also causes a shock to the majority of countries because of the interconnected market (Karkowska & Urjasz, 2021). The Indian stock market has thus far been resilient amidst the COVID-19 crosswind, despite the disrupting waves of the pandemic. According to Dhall & Singh (2020), the COVID-19 pandemic has induced herding behaviour at the industry level. The Indian economy witnessed a subsequent global crash to some extent. A study by Huang et al. (2020) concludes that foreign investors significantly increase crash risk in groups with low levels of real earnings management, while they have no significant effect on crash risk in groups with high levels of real earnings management. In India, it is very difficult to pinpoint the exact impact of the financial crisis, but it seems that the crisis has spilled over into some sectors. Recently, global regulatory lockdowns caused by COVID-19 have severely impacted both the real and financial sectors. In India, shock transmission has substantially increased, resulting in increased volatility. Fractionally integrated generalized autoregressive conditional heteroskedasticity (FIGARCH) has been applied to predict the persistence of the volatility of the Indian indices (NSE). A major goal of this study is to understand how one volatility index’s shocks can affect another’s volatility index.\n\nThis paper has the following structure. The literature review is in Section 2. The data and methodologies are presented in Section 3. In Section 4, we present our results. In Section 5, we summarize the paper.\n\n\n2. Literature review\n\nRecent studies have investigated the cointegration relationship of the stock market amid crises (Aggarwal & Raja, 2019, Bouri et al., 2017, Zhang & Wei, 2010). Some studies have explained the causal linkage while in several other studies authors have performed comprehensive analysis by applying the generalized autoregressive conditional heteroskedasticity (GARCH) 1,1 model developed by Bollerslev (1986). Recent developments in the field of behavioural finance have revealed the volatility clustering in the Indian stock market by analysing time series data. A GARCH model is believed to be extremely useful for modelling and forecasting asset return volatility over time (Engle & Patton, 2001).\n\nMaloney & Mulherin (2003) predicted investors’ irrational behaviour using a volatility forecasting model. Fehr & Tyran (2005) observed that a small group of irrational investors affects the aggregate outcome of the market during crashes; a small amount of individual irrationality may lead to large deviations from the aggregate predictions of rational models. Sadorsky (2006) found that for heating oil and natural gas, the TGARCH model (threshold GARCH model) fits well, whereas the GARCH model fits well for crude oil and unleaded gasoline. Alberg et al. (2008) analysed the dynamic nature of returns in terms of serial correlation, asymmetric volatility clustering, and leptokurtic innovation. Liu & Hung (2010) suggested that modelling the asymmetric component is more important than specifying the error distribution for enhancing volatility forecasts of financial returns when fat tails, leptokurtosis and leverage are present. Muthukumaran et al. (2011) argued that the Indian stock market was highly distressed by global financial crunches. Abdalla & Winker (2012) hypothesized that volatility and expected stock returns are positively correlated. Geels (2013) discussed the green growth discourse which resulted in a major green stimulus program, but this positive effect seems to be coming to an end. Lim & Sek (2013) analysed stock market volatility in Malaysia, finding that the GARCH model works well during a crisis while the TGARCH model works well in the post-crisis period. Asgharian et al. (2013) added the first principal component to the model, which outperformed all other specifications, demonstrating that the constructed principle component is a solid proxy for the economic cycle. Mahalingam & Selvam (2014), amid crises, hypothesized two different sample periods for Bombay stock exchange (BSE) index returns and found that more than 90% of the data were influenced by past values. Danso & Adomako (2014) identified the capital structure of firms in South Africa and found that Africa was not isolated from the impact of the 2007-2008 financial crises. Ding et al. (2014) demonstrated that the influence of the investor’s sentiment trend on stock returns build upon the direction of the investor’s sentiments change (optimistic or pessimistic). Labuschagne et al. (2015) argued that GARCH models produce more accurate results than risk-neutral historic distribution (RNHD) models for constant interest rates. Nowhere in the existing literature has it been mentioned that investors also act rationally during a crisis. Mamun et al. (2015) concluded that the investors have a greed of return, annoyance, and anger; again they are able to evaluate and take all of these behavioural emotions as well as certain key rational attributes in terms of their risk appetite. Bir et al. (2015) examined the volatility shocks in series, and found that volatility clusters are evident in empirical results. Balcilar et al. (2015) suggested that the causal relationships between oil prices are strong. During certain subperiods, but not all, both variables have predictive potential for each other. Molnár (2016) converted a GARCH (1,1) model to a range-GARCH (1,1) model. On 30 shares and six stock indices as well as simulated data, the range-GARCH model outperforms the standard GARCH model, both in terms of in-sample fit and out-of-sample forecasting. Akinsomi et al. (2018) observed the shifting of investors from anti-herding behaviour within the highly volatile market to herding behaviour within the low volatile market. An et al. (2018) found that firms located in countries with higher individualism have a higher stock price crash risk. Using GARCH, Wang et al. (2018) examined how the spillover effect varies over time. Singh (2019) studied the presence of the Monday effect in fear sentiments for all currency pairs, denoting high positive returns with substantial value, and the Friday effect displaying negative returns. GARCH and TGARCH (Ben et al., 2019) were used to analyse asymmetric volatility dynamics in major cryptocurrency markets. The conditional volatilities of equity indices show widespread evidence of asymmetry, structural changes spread to other markets with a big order of magnitude (Harris et al., 2019). Hsu et al. (2020) suggested that negative stock-bond return correlations resulting from investor flight-to-safety become less prominent when stock market uncertainty indices are extremely high. Some studies also identified that the long-term volatility of the stock market depends upon many macroeconomic variables. (Fang et al., 2020) and Lyócsa & Molnár (2020) applied a nonlinear autoregressive model in which the autoregressive coefficient is determined by typical Google searches related to COVID-19 and observed volatility between November 2019 and May 2020. Previous studies (Fang et al., 2020) revealed that the autoregressive coefficient was negative throughout the whole event time; however, as market uncertainty and attention to the virus increased, the coefficient’s magnitude increased as well. Narasimha & Mushinada (2020) point out that investors are concerned about cognitive biases and therefore adapt to changing market dynamics. Vo (2020) investigated the strong positive correlation between foreign investors and crash risk due to asymmetric information in emerging markets. Among BRICS countries (Brazil, Russia, India, China, and South Africa) a diverse response to stock market volatility has been reported including negative and positive shocks (Salisu & Gupta, 2021). Cui & Zhang (2020) suggested that negative information creates fear among investors, which leads to a larger stock price crash risk. Nikkinen & Peltomäki (2020) investigated the investors’ crash fears by analysing the published news on stock market shocks and developing complex associations between information and stock market returns. He et al. (2020) examined the ability of futures markets to price discoveries through margin trading in the stock market. Kumar & Misra (2020) found widespread evidence of long-term similarity between the NIFTY 50 index and the global market. Naik et al. (2020a) examined the robustness of GARCH for both heteroskedasticity and volatility clustering. Elyasiani et al. (2021) analysed market greed by incorporating the skewness index of investing, which captures investor excitement more than investor fear. Engle & Patton (2007) determined that the GARCH model allows long-term volatility predictions to be reliant on socioeconomic dynamics and provides estimates of volatility to be expected in a freshly launched market. Ghani et al. (2022) suggested that the economic policy uncertainty index has a predictive power to forecast. Even during COVID-19, numerous researchers applied the GARCH model; nonetheless, the hedging strategy was expensive, with oil providing maximum hedging effectiveness for Hong Kong (Mensi et al., 2022).\n\nPrevious studies have almost exclusively examined the integration of Asian economies with other developed countries such as the United States and Japan amidst crises. It was reported in the literature (Salisu & Akanni, 2020) that during the crisis, the domino effects hit economies worldwide in the short-run and long-run. This is inconsistent with the arguments given by Aggarwal & Raja (2019), Rajwani & Mukherjee (2013) and Menon et al. (2009).\n\nAlthough most of the current economic crisis has passed, further studies are still required to address developed and emerging Asian markets. To assess the persistence of volatility the FIGARCH model is applied to the Indian stock market. It is necessary to investigate whether the effect persists over time and, if so, for how long? Does the variance of the forecast error in one market change due to a shock in another market? Contributing to existing theory and strategic financial decision-making for investors, this study offers valuable insights. Since the economy is expected to grow rapidly and foreign investors are becoming increasingly interested in the country, it is imperative to understand how market volatility in India varies over time, persists, and is predictable. This may be useful in order to formulate hedging strategies and diversify international portfolios.\n\n\n3. Methods\n\nIn our study, we used data from the NSE-NIFTY 50 (National Stock Exchange) indices. We collected data from two websites1 for the long run; covering most of the recession from January 1, 2008 to December 2, 2021. The period of study is based on the daily return. We calculated the daily return by applying the [Return = log (The closing price of indices/Closing price of indices (-1))] equation to the closing price. We analysed the data using the EViews 12 software package.\n\nH0: Error variance has homoscedastic properties (no ARCH effect) as followed by Lim & Sek (2013), Donadelli et al. (2017), Naik et al. (2020b), Chowdhury et al. (2020) and Dai et al. (2021).\n\nH1: Error variance is heteroscedastic (ARCH effects).\n\nThe autoregressive Conditional Heteroscedasticity (ARCH)2 model of volatility explains that heteroscedasticity may be autocorrelated over time. Conditional informs that variance depends on errors made in the past; heteroscedasticity means unequal variance. This model was proposed by Noble Prize winners (Engle et al., 1987). Suppose that the variance is yt. The model is conditional for the variance yt on yt-1, thus\n\nWe have included the α0 ≥ zero (0) and α1 ≥ zero (0) to remove the (-) variance. In a series if the mean is equal to zero (this can be achieved by centring), then the model can be expressed as follows:\n\nIn ARCH (1) model equation (2) yt2hastheAR1 model\n\n(a) A causal model can only be transformed into a legitimate infinite order MA only when α12<13\n\n(b) yt is white noise when 0≤α1≤1.\n\nARCH (m) process variance at a time is dependent upon observations at previous m times.\n\n\n\nIn theory, yt series squared will be AR (m) with certain constraints applied to coefficients. GARCH models use past squared observations and past variances to calculate variances over time. The model (GARCH 1,1) can be defined as\n\nCovariance stationery observed with the GARCH (1,1) model, this is α1 + β1 < 1.\n\nThe leverage effect predicts that an asset’s price will become more volatile when its price decreases. In response to ‘bad news’, volatility tends to rise, and volatility tends to fall in response to ‘good news’. This is due to financial and operating leverage (Nelson, 1991). A simple variance specification of exponential GARCH is given by:\n\nThe logarithmic form of the conditional variance implies that the leverage effect is exponential and that forecasts of variance are not negative. This hypothesis can be tested to determine whether there is a leverage effect. γ > 0 If γ ≠ 0, then the impact is asymmetric.\n\nFurthermore, the TGARCH model was introduced by Zakoian (1994). The conditional variance of stock market index returns is based on the assumption that unexpected changes in index returns have different effects on the conditional variance of the index returns. Threshold GARCH is a combination of ARCH and GARCH models. It specifies the conditional variance as follows:\n\nwhere dt = 1 if εt < 0 and dt = 0.\n\nIn this model, the good news (εt > 0) and bad news (εt < 0) have differential effects on the conditional variance. Good news has an impact a, while bad news has an impact a + γ it. If γ > 0 then the leverage effect exists and bad news increases volatility, while if γ ≠ 0 the news impact is asymmetric.\n\nThe FIGARCH model modifies this specification by incorporating a fractional difference term (Baillie & Morana, 2009). This variance can be expressed as:\n\nThe NSE shows large shifts during times of crisis, followed by large shifts in the opposite direction, representing the wild and calm periods of volatility clustering. Figure 1 displays a clustering of volatility for NSE returns on a daily basis, as demonstrated by Mandelbrot & Taylor (1967) where “large changes tend to be followed by large changes, of either sign, and small changes tend to be followed by small changes.”\n\nNSE: National Stock Exchange; RNSE: returns of NSE.\n\nFigure 2 illustrates that leptokurtic statistical distributions with kurtosis larger than three result in a greater degree of stock market volatility. While the daily return of the NSE’s Jarque-Bera (32031.42) measures the market’s high volatility, the form of the curve, the magnitude of kurtosis, and the low probability value suggest that it may be possible to reject the null hypothesis.\n\nNSE: National Stock Exchange; RNSE: returns of NSE.\n\nIn Table 1, the least square method is used to identify correlations between the dependent variable and independent variables by combining the mean and median, and the GARCH model is used to calculate the error distribution.\n\nTable 2 shows that the statistics (73.209) and probability value (0.00) are statistically significant for the presence of ARCH effects. It is also estimated the value of α1=0.145, this indicates that the null hypothesis has been rejected. The heteroscedasticity test confirmed the existence of ARCH effects in the Indian stock market.\n\nTable 3 reveals the variance equation hence the GARCH (1,1) model is justified for the presence of time-varying conditional volatility of NSE returns.\n\nThe mean equation from Table 3 can be derived as:\n\nAt present, the average return of the NSE is 0.000691 and its past value significantly forecasts the current series by 0.0601.\n\nThe coefficients were positive and statistically significant. We obtained the following variance equation for the NSE return:\n\nFor the long term with constant variances, the GARCH and ARCH parameters are statistically significant at the 1% level (Table 3). The results of the GARCH model are as follows. The time-varying volatility of NSE daily returns includes a constant (0.00000175), past errors (0.893), and a component that depends upon past errors (0.098).\n\nThe GARCH (1,1) model and ARCH parameters indicate the persistence of volatility shocks. As a consequence, today’s shock is implied to remain in the forecast for years to come. In addition, we also examined the long-term dynamics of the Indian stock market using the FIGARCH. The lagged volatility and fitted variance are confirmed from the estimation output, and when comparing Table 3 and Table 4 GARCH and FIGARCH, we can see that the ARCH coefficient increases away from 1.00 whereas the GARCH coefficient decreases away from 1.00. This result highlights the long-term persistence of volatility shock in the Indian stock markets. Moreover, we analysed the impact of good and bad news on the volatility of the Indian stock market using the TGARCH and exponential GARCH (EGARCH) models. The multiplicative dummy variable (Table 5) was added to the GARCH model to identify statistically significant differences when the shocks were negative.\n\nNevertheless, the volatility behaviour of market index returns varies across market stages, and the Indian stock market has undergone various stages of development. We estimate the time varying volatility of positive shock; σt2= 0.00000377 + 0.874 + (-0.001).\n\nSimilarly, we estimate the time varying volatility of negative shock\n\nThe difference between good and bad news in the NSE index is 0.193 which is the coefficient of the asymmetric term. The coefficient of the asymmetric term is negative (Table 6) and statistically significant at 1% level. In exponential terms this indicates that bad news has a greater effect on the volatility of NSE than good news.\n\nAs previously discussed, the basic GARCH model assumes that positive and negative shocks of the same absolute magnitude have the same impact on future conditional variances in the Indian stock markets. In contrast, previous studies (Alberg et al., 2008, Labuschagne et al., 2015, Mathur et al., 2016) have shown that the volatility of aggregate equity index returns can respond asymmetrically to past negative and positive returns, with poor returns resulting in higher volatility in the future. In economics, this is often referred to as the “leverage effect.”\n\n\n4. Results\n\nFrom the above analyses it is clear that, regardless of the fit effect or estimation accuracy, GARCH models can be appropriately adapted to the volatility of the Indian stock market. Furthermore, GARCH (1,1, symmetric model), TGARCH and EGARCH (asymmetric models), perform well in our out-of-sample estimation. These empirical results can generally be categorized into two sections, beginning with the ARCH, GARCH, and FIGARCH models followed by an analysis of the TGARCH and EGARCH models using their main objectives. The preliminary analysis of the NSE indices is based on the analysis of different descriptive statistics. Table 3 demonstrates significant coefficients for constant variance, ARCH, and GARCH parameters at the 1% level. These results pertain to the GARCH heteroscedasticity model. The constant (0.00000175) was coupled with its past (0.893) and past errors (0.0.098). Our findings are consistent with Mathur et al. (2016). These parameters also indicate resilience of volatility shocks. Based on the estimated output, lagged volatility and fitted variance are significant. Figure 3 indicates that long-term forecast periods are associated with greater uncertainty, and short-term forecast periods are associated with lower uncertainty. Based on this study, investors can select companies in accordance with their risk aversion. The conditional volatility of the market return series from January 2008 to December 2021 shows volatility shifting across time, with violent price swings clustering around the boom. Higher prices emerged in response to solid economic fundamentals, but the real cause appears to be imperfections in the Indian market.\n\nGARCH: generalized autoregressive conditional heteroskedasticity; NSE: National Stock Exchange; RNSE: returns of National Stock Exchange.\n\nWhen comparing the findings produced using GARCH and FIGARCH, as shown in Table 4, the ARCH coefficient increases away from 1.00 whereas the GARCH coefficient falls away from 1.00. The consequences of current shocks are evident in the variance prediction for subsequent years. The difference between positive and negative news in the NSE index is 0.193, which is the asymmetric term’s coefficient. Our research revealed that the volatility of the Indian stock market could be affected asymmetrically by recent negative and positive returns, with a particularly negative rate of return resulting in greater future volatility.\n\n\n5. Conclusion\n\nThe heteroscedasticity test confirms that there is an arch effect in the Indian stock market. Thus, the GARCH (1,1) model is justified for time-varying conditional volatility of NSE returns. As shown in Table 4, the mean equation is r_nse = 0.000691 + 0.060154. This study is based on secondary data for a period of 13 years ranging from January 2008 to December 2021.The GARCH (1,1), FIGARCH and EGARCH approaches are applied to determine the long-term persistence of volatility. Based on the results, the null hypothesis is rejected. The returns on stocks appear to be stable, though very volatile. As a result of the current shocks, future forecasts of variance are likely to be affected for several years. Information, news, and events can also significantly impact the stocks’ volatility. We observed an asymmetrical reaction in the NSE return series in response to both good and bad news. Due to the leverage effect, a negative innovation (news) would have a greater impact on volatility than a positive innovation (news). According to this stylized fact, the innovation sign significantly affects the volatility of returns and bad news increases volatility more than good news. Therefore, we conclude that bad news in the Indian stock market increases volatility more than good news. Volatility models derived from the GARCH equation provide accurate forecasts and are useful for portfolio allocation, performance measurement, and option valuation.\n\nMoreover, it would be useful to investigate the volatility forecasting and impact of good and bad news on the inclusion of a larger sample of countries from Asia, Africa, North America and Europe in comparison to the pre- and post-crisis periods.\n\n\nData availability\n\nFigshare: Modelling time-varying volatility using GARCH models. https://doi.org/10.6084/m9.figshare.20681203.v2 (Ali et al., 2022)\n\nThis project contains the following underlying data:\n\n• Data.xlsx (data from the NSE-NIFTY 50 [National Stock Exchange] indices)\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).",
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Publisher Full Text\n\nVo XV: Foreign Investors and Stock Price Crash Risk: Evidence from Vietnam. Int. Rev. Financ. 2020; 20(4): 993–1004. Publisher Full Text\n\nWang Y, Pan Z, Wu C: Volatility spillover from the US to international stock markets: A heterogeneous volatility spillover GARCH model. J. Forecast. 2018; 37(3): 385–400. Publisher Full Text\n\nZakoian JM: Threshold heteroskedastic models. J. Econ. Dyn. Control. 1994; 18(5): 931–955. Publisher Full Text\n\nZhang YJ, Wei YM: The crude oil market and the gold market: Evidence for cointegration, causality and price discovery. Res. Policy. 2010; 35(3): 168–177. Publisher Full Text\n\n\nFootnotes\n\n1 The different websites used to collect the data of Indices are www.nse.com and www.yahoofinance.com.\n\n2 The generalized autoregressive conditional heteroskedasticity (GARCH) process was introduced by economist and Nobel Memorial Prize winner Robert F. Engle in 1982. The approach is intended to estimate financial markets’ volatility."
}
|
[
{
"id": "151678",
"date": "10 Oct 2022",
"name": "Diksha Panwar",
"expertise": [
"Reviewer Expertise Operations",
"Digital Marketing",
"Financial Marketing. Modelling"
],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe theme is intriguing, and I can see value in research: - “Modelling time-varying volatility using GARCH models: evidence from the Indian stock market”- Forecasting the volatility of the Indian stock market has become essential for economic and financial analysts as a result of the financial crisis. An analysis of the NSE index returns showed that the Indian stock market had an autoregressive conditional heteroskedastic trend. The author of this paper has looked into the Indian stock market's volatility from 2008 through 2021. The GARCH equation, which is helpful for portfolio allocation, performance evaluation, and option valuation, has been utilised by them to produce precise projections.\nThe introduction is well written and constructed extremely nicely: - The introduction is so well constructed as it grabs the interest of the reader, as well as it was clear, brief, purposeful, and focused.\nThe presentation of the result and discussion part has been well-organised: - The GARCH model, which is properly fitted to the volatility of the Indian stock market, was employed by the author for this study. If we take a closer look, the study may be divided into two sections: an investigation of the TGARCH and EGARCH models based on their primary goals, and then the ARCH, GARCH, and FIGARCH models. Based on the examination of various descriptive statistics, the NSE indices' preliminary study. Lagged volatility and fitted variance are substantial based on the estimated output. If we dig deeper into the data, we find that longer prediction durations are linked to higher levels of uncertainty whereas shorter forecast periods are linked to lower levels of uncertainty. According to this study, investors can choose companies based on how risky they are. The potential instability of the market return series from January 2008 to December 2021 shows volatility shifting across time, with violent price swings clustering around the boom.\n\nI certify that I have reviewed this work and possess the requisite level of knowledge to testify to its scientific quality.\n*Further research can be conducted using big data analytics techniques to get more accurate forecasting of Indian stock market*\n\nIs the work 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": "157105",
"date": "02 Dec 2022",
"name": "Suleman Sarwar",
"expertise": [
"Reviewer Expertise My area of expertise are financial economics",
"portfolio analysis",
"energy economics and environmental economics etc."
],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 Comments to authors:\nIntroduction:\n1. There are a few sentences which need references, such as “In times of crisis, investors make errors of judgment as long as a group of investors make irrational decisions, leading to the worsening of the situation in the stock market.”\n2. In the introduction, the author has to clearly mention the main contribution of this study. See for example1,2.\nLiterature:\n3. I suggest that author has to write the literature in a systematic way,\n4. I recommend some of the recent studies need to be included in literature, such as 2019, 2020 etc.3,4,5,6,7.\nData and Results:\nIf we focus on results section, it needs modifications.\n5. Why is it important to discuss this Indian Market instead of other markets?\nConclusion:\n6. First paragraph of conclusion is too lengthy, please report the main findings and specific (instead of general) policy implications.\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": []
}
] | 1
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https://f1000research.com/articles/11-1098
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https://f1000research.com/articles/11-1455/v1
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08 Dec 22
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{
"type": "Research Article",
"title": "Japanese international student-athletes’ adjustment experience at the National Collegiate Athletic Association",
"authors": [
"Hirokazu Matsuo",
"Takuya Tsukamoto",
"Haruka Kasahara",
"Kohei Funasaki",
"Ryu Sakamoto",
"Motohiro Fujino",
"Takuya Tsukamoto",
"Haruka Kasahara",
"Kohei Funasaki",
"Ryu Sakamoto",
"Motohiro Fujino"
],
"abstract": "Currently, the number of International-Student Athletes (ISAs) in the National Collegiate Athletic Association (NCAA) is increasing over time and Japan is the biggest market for recruiting in Asia. Whereas international students are playing an increasingly important role in the NCAA, several studies have reported that ISAs face challenges in adjusting to their dual identities as students and athletes at universities in the US. Furthermore, it has been cleared Asian students studying at US universities have some challenges because of cultural and linguistic differences. However, it remains unclear whether the difficulties experienced by Japanese ISAs are the same as or different to the adjustment-related challenges experienced by other ISAs or Asian students. The purpose of this study is to clarify challenges for ISAs from Japan in adjusting to the United States’ universities. Situating in the ISA adjustment model and using a unique case study design, 13 Japanese ISAs at the NCAA Division I universities were interviewed, and data were coded by a continuous comparative analysis method. The results revealed that Japanese ISAs who participated in this study faced academic, social, athletic, personal-emotional, and institutional adjustment challenges. Particularly, Japanese ISAs differed from ISAs from other countries in several points; cultural differences, differences in communication styles, and systemic differences in academia and athletics between Japan and the US. For promoting adjustment of Japanese ISAs, this study suggests; gaining experience and getting information to understand and familiarize with the differences, building relationships with linguistic and culturally diverse people to understand various cultures, encouraging universities and coaches to understand specific difficulties for Japanese ISAs.",
"keywords": [
"Inter-collegiate athletics",
"Cross-cultural adjustment",
"Cultural transition",
"Cultural adaptation",
"Academic sojourner",
"Mental health"
],
"content": "Introduction\n\nOne of the main activities of university coaches in the United States (US) is recruiting students, and the talent they acquire often determines the success or failure of their teams. Many coaches, especially at the Division I (DI) level in the National Collegiate Athletic Association (NCAA), are required to find talented athletes in a wide range of regions. Therefore, the proportion of their total budgets spent on recruitment is reported to be increasing over time (Judson et al., 2004; Sander, 2008). In addition, international students are playing an increasingly important role in the NCAA because of the growing trend to recruit student-athletes from outside the US (Drape, 2006). US universities recruit student-athletes globally because the number of international student-athletes (ISAs) is increasing over time. ISAs are international students from other countries who play sports on university varsity teams as student-athletes.\n\nA total of 17,653 ISAs were reported to have participated in the NCAA in 2009–2010, which is a significant increase from just under 6,000 a decade earlier (Erin et al., 2010). In 2021, 21,334 ISAs played in the NCAA, indicating a further increase over the past decade (NCAA, 2021). The number of ISAs in DI in 2021 was 1.55 times higher than the number in 2012 (NCAA, 2021), suggesting that coaches will continue to consider ISAs as an essential component of their teams in the future. The following countries have more than doubled the number of ISAs playing in the NCAA since 2015, and had 100 or more ISAs in 2021: Italy, Portugal, the Netherlands, Iceland, and Greece in Europe; Argentina in South America; Kenya and Tanzania in Africa; and Japan in Asia (NCAA, 2021). These factors suggest that Japan is an important market when considering the recruitment of talented student-athletes from Asia.\n\nHowever, several studies have reported that ISAs face challenges in adjusting to their dual identities as students and athletes at US universities (Ridinger and Pastore, 2000a; Sato & Hodge, 2009; Popp et al., 2010; Pierce et al., 2011). In addition, it has been reported that the challenges faced by Asian students studying at US universities include academic, social, and language difficulties caused by cultural and linguistic differences (Sato & Hodge, 2009). Sato and Hodge (2015) reported that Japanese students studying at US universities experienced academic and social difficulties that were created and exacerbated by cultural and language differences, collectivist social patterns, and negative experiences with professors and other students. However, it remains unclear whether the difficulties experienced by Japanese ISAs are the same or different to the adjustment-related challenges experienced by other ISAs. Thus, university coaches may be unaware of Japanese ISA-specific challenges when recruiting ISAs from Japan. Therefore, the current study aimed to elucidate this situation for ISAs from Japan, which is likely to become an increasingly critical recruiting market for US universities in the future, by identifying the challenges of university adjustment among Japanese ISAs. To address this issue, the study focused on the following research question: What challenges are faced by Japanese ISA in adjustment to a US university?\n\n\nLiterature review\n\nIt has been suggested that the adjustment process of ISAs differs from that of other international students (Ridinger, 1998). Popp et al. (2009) investigated international and domestic student athletes’ adjustment to US universities and their differing views regarding the purpose of college sports. The results revealed that international students were less socially adjusted and less likely to belong to an organization than domestic students. Although the literature on ISA experiences at US universities is mixed, with both positive and negative findings (Connell, 2007), many studies have shown that ISAs face academic and social difficulties at US universities. In particular, ISAs whose native language is not English may struggle to meet the NCAA’s academic eligibility requirements (Connell, 2007).\n\nPierce et al. (2011) expanded on Ridinger and Pastore’s (2000b) preliminary study of ISAs to examine the most significant challenges faced in the first year of college by surveying a diverse population. In that study, 355 student-athletes from 15 NCAA Division I schools responded to the survey, 192 of which were from countries other than the US. The results revealed that homesickness, adjustment to US culture, and language were the three most common concerns of ISAs (Ridinger & Pastore, 2000b). Andrade (2005) examined the transitional issues unique to international students during their first year at a US university. Many of the challenges faced by international students are related to adjusting to a new environment, leaving home for the first time, developing effective study habits, and choosing a major. While some of these overlap with domestic students from the US, these potential challenges are magnified by language barriers and cultural differences for international students. Comeaux and Harrison (2011) reported that striking the right balance between academic and athletic life can be challenging for student-athletes in transition, who must constantly negotiate the dual roles of student and athlete. They suggest that these competing roles, coupled with time demands, can interfere with academic and athletic performance.\n\n\nTheoretical framework\n\nBefore proceeding with the theoretical framework, we define cross-cultural adjustment. Palthe (2004) defines cross-cultural adjustment as “the process of adaptation to living and working in a foreign culture.” Furthermore, adjustment includes the degree of comfort and familiarity that is perceived by migrants in the new country (Black et al., 1999). Because the theme of the current study was the adjustment of Japanese ISAs to US universities, we used the ISA adjustment model developed by Ridinger and Pastore (2000a) and extended by Popp et al. (2010) as the basis for our study. Ridinger and Pastore (2000b) investigated the adjustment of ISAs and general international students to US universities and found that ISA students were better adapted to their new environment compared with the general population of international students.\n\nRidinger and Pastore (2000a) developed the ISA adjustment model that has included (a) antecedent of adjustment, (b) adjustment task, and (c) outcome. (a) The antecedent of adjustment is personal, perceptional, cultural distance, and interpersonal factors. The four factors directly influence the (c) ISA outcomes (i.e., satisfaction, academic performance, and athletic performance). These antecedents and outcomes relationships are influenced by (b) adjustment tasks (i.e., academic, social, athletic, personal-emotional, and institutional attachment).\n\nPrevious studies examined the detail of four antecedent factors. Ridinger and Pastore (2000a) identified “self-efficacy” and “technical competencies” (e.g., athletic, academic, and English language skills) as personal factors of ISAs. Furthermore, Popp et al. (2010) added “travel experience” and “adventure” as personal factors. Perceptual factors included ISA’s “realistic expectations” regarding both their chosen college and athletic discipline, as well as the material and psychological “social support” provided by the college and athletic departments, while “family influence” was added by Popp et al. (2010). Cultural distance refers to differences between the indigenous culture of the ISA and campus culture, including physical and geographic distance and social and customary differences. Finally, in terms of interpersonal factors, ISAs reported that good relationships with “teammates,” “coaches,” and “faculty and staff” promote adjustment (Popp et al., 2010). However, Popp et al. (2010) reported that relationships with “faculty and staff” did not promote adjustment in ISAs.\n\nThis theoretical framework describes the unique adjustment challenges of ISAs from different social and cultural backgrounds and the factors that influence their adjustment. Because academic, social, athletic, personal-emotional, and institutional attachments are expected to be experienced regardless of ISAs’ country of origin, it is possible to apply the same framework to Japanese ISAs. However, Japanese students have great difficulty with cultural and language differences, collectivist tendencies, negative experiences with professors and other students, and building social relationships with white students. This tendency may have led to differences in their adjustment experiences with the ISA. Therefore, this study adds to the literature related to the adjustment experience among ISAs and provides essential insights into the adjustment of Japanese ISAs to decision-makers, including the Japanese ISA community and universities, while filling a gap in the literature.\n\n\nMethods\n\nThis study used a unique case study design (Yin, 2003) focusing on the challenges faced by Japanese ISAs in adapting to a US university, and a single case study (Patton, 2002). The purpose of a case study is to better understand a complex educational and social phenomenon, while at the same time retaining meaningful specificity in a real-world context (Yin, 2003).\n\nPurposeful sampling (Patton, 2002) was chosen for this study to gather a wealth of information regarding the research question. The selection of participants for the case study was conducted by contacting student-athletes who met the selection criteria for inclusion and directly inviting them to participate in the study. Because of the limited number of Japanese ISAs in DI at the NCAA who were eligible, we followed the findings of Guest et al. (2006) and aimed to recruit a minimum of 12 participants. The target group consisted of 13 individuals (six males and seven females) who met the following selection criteria and volunteered to participate in the study: (1) Japanese citizenship, (2) Japanese primary and secondary education, and (3) at least 1 year of experience as an ISA in the NCAA’s DI program. These participants had received Japanese education from childhood. All participants were considered to have experienced the adjustment issues described by Connell (2007) and Comeaux and Harrison (2011) when they entered US universities.\n\nThe participants were 13 Japanese ISAs who volunteered for the study. There were no minors among the subjects. All participants were adults who have Japanese nationality. Nine enrolled in college during the study period, and four had already graduated. Six were male, and seven were female. Four played soccer, three played golf, two played American football, two played ice hockey, one played basketball, and one played tennis.\n\nThe authors were accepted to conduct this study from the institutional ethics committee of the University of Tsukuba (Tai 021-82). Many of the subjects were living in the U.S., and it was difficult to receive their signatures in writing in person due to the geographical distance from Japan, where the researchers are located, and the restrictions on travel caused by the COVID-19 pandemic. Therefore, we retain the video recording of the verbal consent and the documentation of the content and date of the informed consent. This informed consent process was approved by the ethics committee of the university.\n\nThe data collection method consisted of face-to-face semi-structured interviews with each participant to obtain information about the challenges they faced in adjusting to college as Japanese ISAs. The research ethics committee at the University of Tsukuba approved the study before the interviews were conducted.\n\nEach participant was interviewed using a semi-structured interview technique (Yin, 2003). Specifically, we used an interview guide consisting of 17 questions about the challenges the participants experienced in adjusting to college as Japanese ISAs. The questions primarily asked about experiences related to academic, social, athletic, personal-emotional and school institutional adjustment in college. The questions were developed on the basis of previous research (Ridinger & Pastore, 2000a; Popp et al., 2010). Participants were interviewed using an online conferencing system, and interviews typically lasted 60 to 90 minutes. Interviews were conducted for each participant between July 2 and July 16, 2021. As needed, follow-up questions to clarify participants’ comments were asked by phone (Shuy, 2002) or email (Meho, 2006). After this process, the researcher transcribed the interviews, returned the transcribed data to all participants, and conducted member checks (Lincoln & Guba, 1985; Patton, 2002) via email (Meho, 2006). This process ensured that the transcribed data accurately represented the participants’ views and experiences of adjusting to college. The full interview guide in Japanese can be found in the Extended data (Matsuo et al., 2022). Table 1 is the list of the questions under which the themes arose.\n\nThe purpose of triangulation is to assess the accuracy of the data rather than to seek universal truths (Merriam, 1998). Triangulation used multiple perspectives, including data from interviews, which were interpreted through the ISA’s adaptive model lens. Member checks were conducted to mitigate the effects of subjective bias (Patton, 2002), and copies of the transcribed interview data were emailed to individual participants. Participants confirmed the accuracy of the data and the researcher’s interpretation of the data to ensure that reliability had been established (Merriam, 1998).\n\nPeer debriefing was conducted by two qualitative researchers with expertise and experience in this area, who determined that the interpretation of the data was accurate and representative of the participants’ statements. A continuous comparative analysis method (Klapwijk & Boeije, 2010) was used to interpret the data, enabling a comparison of different pieces of data in detail. Specifically, the first and second authors independently coded potentially meaningful data from each participant’s interview transcript. Any differences were discussed until consensus was reached. Two additional peer debriefers then checked the codes to avoid researcher bias. The coded data from each participant’s records were compared to identify similarities and differences. All data and keyword definitions were returned to participants for a second member check. After receiving final confirmation from all participants, the researcher grouped the codes into thematic categories, then narrowed them down into recurring themes (Klapwijk & Boeije, 2010).\n\n\nResults\n\nFive major themes emerged from the data that corresponded to and were interrelated with the themes reported by Popp et al. (2010): (a) academic adjustment challenges, (b) social adjustment challenges, (c) athletic adjustment challenges, (d) personal-emotional adjustment challenges, and (e) institutional attachment challenges. These themes are described below in narrative form, with quotes from participants.\n\nThis theme represents participants’ difficulties with academic adjustment related to coursework, classes, and assignments. Ten participants experienced language barriers related to their academic work and had difficulty understanding the course content in English and speaking in class. For example, Kusanagi explained that. The findings are reported using pseudonyms to disguise the identities of participants.\n\nWhen I went to class, I did not know what the professor was talking about. Then, after the class was over, there was an assignment for each class, but I didn’t know what the assignment was. If I couldn’t do the assignment, I wouldn’t get any points for that class, and my grades would drop. I had a lot of trouble with that in the first year.\n\nKusanagi reported that his English skills were not good enough to understand what the professor said in class, or to comprehend the assignments. He had difficulty coping with classes and assignments in an unfamiliar language. Another participant, Nagase, described her difficulties with speaking English in class. She stated that she lacked confidence in her English because she was not a native speaker, which acted as a psychological barrier and made her hesitant to speak up.\n\nI couldn’t keep up with the discussion because of the fast pace at which everyone was speaking, and because I wasn’t sure if what I was doing was correct. So, I wasn’t confident enough to say what I was doing. I guess the problem was less about the content of the discussion.\n\nNagase had particular difficulty participating in group discussions and seemed unable to speak her mind with confidence. Furthermore, four of the participants mentioned cultural differences in terms of academic studies. For example, Ohishi was perplexed by the difference between the teaching style at the US university and that in Japanese school classes.\n\nI think the content and style of classes are quite different (from Japan), aren’t they? It depends on the class, but in my first year, about half of the classes were in large classrooms and half were in small groups, and there were difficulties in both types of classes. In smaller classes, there was inevitably more communication and more group discussions. It was difficult for me to participate actively in those situations.\n\nAfter years of experience with the one-way lecture teaching style in Japan, Ohishi noted that he had difficulty adapting to the small-group, discussion-based teaching style at the US university.\n\nThis theme represents participants’ difficulties in social adjustment in building relationships. In particular, nine participants were confronted with social and cultural differences, and experienced difficulties regarding differences in background experiences, perceptions, and communication styles. For example, Sugimoto explains.\n\nThe biggest challenge I faced was cultural differences. My teammates were from eight different countries, so each of us had a different culture and lifestyle, and it took me a long time to understand them. To be more specific, the way we studied was different. Japanese people are serious and do their assignments three days to a week in advance, but some other student-athletes have the option of not doing their assignments.\n\nSugimoto stated that cultural differences were the biggest challenge she faced. She felt bewildered by the differences from Japanese customs and mindset when building relationships with teammates from diverse backgrounds. Moreover, she felt stressed by this gap. In contrast, Matayoshi reported that differences in communication styles were challenging to adapt to.\n\nJapanese people, I think, are very considerate. Even if they do not say everything that is on their mind, they can read the mood of the situation. But with Americans, for example, if you do not express your opinion, they take that to mean that you don’t think about anything. So, I have to express my opinion.\n\nMatayoshi found it challenging to transition from the Japanese-specific communication style he had experienced during his life in Japan to the frank, opinionated communication style required in the US. In addition, the fact that they could not communicate everything they wanted to say in English made communication even more difficult. In addition, seven of the participants mentioned building relationships. For example, Nagase, as a racial minority on the team, felt uncomfortable in his relationships with his teammates.\n\nThere are differences between people of different races, and, to be honest, ice hockey is a white sport. So when you have a different culture and a different language, there is no one on the team that I have things in common with.\n\nI was pretty confused at first because I felt like there was no one I could understand, even though I was in the same place every day.\n\nNagase described his experience on a predominantly white ice hockey team. She had difficulty socially adjusting because of the lack of teammates with similar cultural backgrounds. As a result, they had difficulty building mutual understanding and respectful relationships. In addition, Yazawa mentioned building relationships with coaches.\n\nMy relationship with the coaches was good. I communicated with the coach, so I had a good relationship with him, and the head coach was a very kind person who talked to me a lot. The only thing is, the scouting team coach seemed to hate Asians, so I was pretty much ignored at the beginning of the season when I started working with the scouting team.\n\nYazawa had a good relationship with his coaches while playing as an American football team member. However, he had experienced being ignored by specific coaches who discriminated against Asians, and he described his experience of being exposed to cultural bias. Five of the participants also mentioned difficulties related to the social language barrier. For example, Yokota explained.\n\nStudents tend to use the latest slang, as in Japan. So when I came here, I thought, “What? I have never heard of that word, and it’s not even in the dictionary. They don’t teach you that at school.\n\nYokota seemed to have difficulty conversing with young people because of the slang they used. Because she had not learned this language in Japan or at language schools, she was confused when slang was used in everyday conversation and she could not understand it.\n\nThis theme represented difficulties with competitive performance, relationships, and competitive adjustments related to conditioning, such as diet and injuries. Eleven participants mentioned their on-field performance, including gaining playing opportunities, gaps in physique and athletic ability, and tactical adjustments. For example, Nagase discussed gaining playing opportunities as follows.\n\nTo be honest, I did not play many games in my junior year. I was on the bench all the time. In terms of playing time, there were times when I did not play in a single game, even in my third year, and, on the other hand, there were times when I played all the time consistently.\n\nNagase seemed to have a hard time with this situation, not only immediately after entering the university, but always needing to compete within the team to win playing opportunities. Inoue also described the gap in physical size and athletic ability as follows.\n\nMy body is small, to begin with. I am of average height for a Japanese person, but I have a skinny frame. When I went to the US, I felt that the physicality was different.\n\nI started to think with my head a lot more, and I became able to read, predict, and think about the game. Eventually, it worked to some extent. Compared with the first year, now I can play soccer the way I want to play.\n\nInoue felt a gap between her physique and athletic ability, which were acceptable when she played in Japan, and those of players in the US. She attempted to adapt to this gap by training her physique and improving her play prediction ability. As a result, she has been able to play successfully in the NCAA. Furthermore, four participants mentioned diet, including diet quality and access to Japanese food, in relation to their competitive adjustment. For example, Nagase stated that.\n\nThe food is not very good. It is not good in terms of taste, and in terms of eating a proper balance of food that suits one’s body. It is a dormitory, so you can eat what you like in a buffet style. Although I tried to take care of myself, there were times when I gained weight or gained more than I should have. I think it’s one of the most challenging aspects of the program.\n\nNagase was not satisfied with the quality of the food provided in the university dormitory and was not eating the kind of food he wanted. Participants who mentioned diet seemed to relate it to athletic adjustments based on their perceived diet as necessary for athletic performance.\n\nThis theme represented difficulties with personal emotional adjustment, and factors related to climate that affect psychological and emotional states. Eight participants mentioned changes in their psychological state, including homesickness, lack of motivation, and loneliness. For example, Sugimoto described homesickness in the following way.\n\nThe first was homesickness. It was the first time I had lived away from my family for an extended period, so I was homesick. I think the stress of cultural differences also caused homesickness.\n\nSugimoto seemed to feel homesick because of the stress caused by cultural differences and living away from his parents for the first time when he enrolled in a US university. In addition, Ohishi described a loss of motivation.\n\nI felt that if I did not do well in soccer, my motivation for the entire study abroad program and classes would decrease.\n\nOhishi felt that soccer was the one sport where he could express himself as a student-athlete. If he did not do well in that sport, it would be difficult to maintain motivation for his entire college life and academic work. In addition, Ando described his sense of loneliness as follows.\n\nAt first it was hard to accept that my teammates’ parents could come to watch the golf matches, but mine could not.\n\nData felt lonely because she was far away from her parents and, like her teammates, her parents could not come to support her in her golf game. This situation occurred because of the geographical distance from Japan, and she had to accept an environment in which she could not receive the support of her parents. Five participants also mentioned the climate, including cold and inclement weather. For example, Nagase described how cold weather was related to personal emotional adjustment.\n\nIt is also cold and dark, especially in winter. So I felt very depressed, especially in winter. Moreover, the weather is not good. In winter, the sun rises late and sets early, so if it snows or the sky is dark, you feel cold, and everyone, not just international students, says that it makes them a little depressed.\n\nNagase described the region’s climate where he attends his university as often being depressing, especially in the winter, with low temperatures and a lot of snow. He also mentioned that this feeling was not limited to international students. Thus, adjustment to the climate appeared to be necessary for ISAs depending on their locations.\n\nThis theme represented difficulties with school institutional adjustments related to NCAA rules and university systems. Five participants mentioned NCAA rules, including training volume, frequency, and eligibility. For example, Yazawa discussed training volume and frequency as follows.\n\nNCAA rules limit the number of days you can practice football. There are only about 30 days in the spring season in the spring, and in the fall, you can only practice in August, 1 month before the season starts in September. Practice with pads is minimal.\n\nI don’t think it is necessary to practice all year round like in Japan, but it would be nice to have a little more practice.\n\nYazawa described the gap between his playing experience in Japan, where it is normal to undertake a lot of training, and the amount and frequency of training required by NCAA rules. Regarding football, Yazawa felt that he did not have enough time to prepare appropriately for a short period between practices, which included contact. Kusanagi also described eligibility as follows.\n\nI think the education system was the most challenging part for me. I knew that in the American educational system, you have to study. If you do not study hard and get good grades, you cannot play soccer. I had to study hard, and it was not easy.\n\nKusanagi mentioned that to maintain eligibility in the NCAA, he needed to obtain sufficient academic grades. This situation is very different from the Japanese educational system, and participants seemed to have difficulty balancing sports and academic studies. In addition, five participants referred to the characteristics and services offered by the university. Sugimoto, for example, described this issue as follows.\n\nThe university I went to only had outdoor tennis courts in its facilities. So if it rained, training was canceled, and we just trained at the gym. Before matches, I would go to the indoor court to practice, which was 30 minutes away by car. It was very inconvenient. It would have been nice to have an indoor court.\n\nSugimoto was not satisfied with the facility he used for tennis practice. However, he had to adapt to the inconvenience of using a different facility, which is not something the individual student can control. Thus, adapting to NCAA regulations and the university itself also challenged the Japanese ISAs.\n\n\nDiscussion\n\nThe main findings of this study indicate that Japanese student-athletes experience challenges in academic, social, athletic, personal-emotional, and institutional adjustment (Popp et al., 2010). A theoretical framework was applied to understand their adjustment experiences. While the participants exhibited similar tendencies to ISAs in general, they appeared to be perplexed by cultural differences between Japan and the US, differences in communication styles, and systemic differences in academic study and athletic training that are unique to the Japanese context. Participants described these challenges by making comparisons with their experiences in Japan when discussing their experiences of the adjustment process.\n\nFirst, in terms of academic adjustment, students appeared to have difficulty expressing their opinions in discussions because of the gap between the lecture-oriented teaching style in Japan and the tendency to conform their opinions to the generally accepted correct answers. Kaczmarek et al. (1994) reported that international students may face some of the same adjustment problems during the transition to higher education as American students. In addition, they noted that international students may have a more difficult transition to college than American students because they are faced with an unfamiliar language and culture in a new country. Among them, Japanese students who studied at US universities found that cultural and language differences, collectivist tendencies, and negative experiences with professors and other students created and exacerbated their academic and social difficulties and feelings of alienation and isolation (Sato & Hodge, 2009). In addition, Japanese school environments tend to emphasize effort, group activities, and peer control, while American schools tend to emphasize competence, individual activities, and teacher control (Stevenson & Stigler, 1992).\n\nFurthermore, according to Allen and Ingulsrud (2005), in Japanese texts, unimportant content is typically located at the top of the page, whereas meaningful content is located at the bottom of the page. In English texts, in contrast, the first sentence typically conveys the paragraph’s theme. As a result, readers must apply different perceptual processes when reading the two languages because of differences in reading patterns (Tomika & Paradis, 2002). Adapting from Japan’s collectivist society to an individualistic society is another challenge for Japanese students studying at US universities, where individualistic educational systems often criticize students who do not speak up in class when teachers pose questions (Hofstede, 2001). In such cases, students who do not participate in classroom discussions are considered to be unable to follow or understand the lecture. According to Hofstede (2001), students in collectivist cultures tend to consider it inappropriate to speak up unless called upon by the professor. This cultural and linguistic background may also make the academic adjustment challenges of Japanese ISAs more difficult.\n\nIn addition, in the task of social adjustment, participants struggled with cultural differences from Japan, including differences in background experiences and perceptions, as well as differences in communication styles. In many cases, participants were particularly stressed by differences in daily habits and mindset. In past studies, minority students at predominantly white colleges and universities have reported difficulty establishing social relationships with their white peers and hesitancy in communicating with their white peers, despite the need for social adjustment and assimilation (Sato & Hodge, 2009). As Barnlund (1989) noted, Japanese students typically do not expose their private selves, including their emotions, in conversations with strangers, and express their problems only to their inner circle of friends and family.\n\nFurthermore, language barriers are among the most common challenges faced by Japanese students when establishing social relationships, causing anxiety, fear, and isolation (Bang et al., 2008). In addition, the speech patterns and tones of other languages are often tied to the native language, and some English sounds are complicated for native Japanese speakers to acquire (Wells, 2000). Regarding differences in communication styles between Japan and the US, it is also necessary to understand the differences between high-context communication and low-context communication. High-context communication tends to be inadequate and implicit, and contains indirect messages, whereas low-context communication contains a large amount of information and is explicit (Hall & Hall, 1990). In other words, in high-context communication, such as that conducted between Japanese people, listeners are expected to guess what the speaker is trying to say with a few statements. These differences in speech can easily lead to misunderstandings when Japanese and American people converse, which can affect individual thoughts and feelings (Barna, 1998).\n\nDifferences in language and cultural backgrounds also appear to impact the challenges of athletic adjustment. Comeaux and Harrison (2011) noted that striking the right balance between academic and athletic life can be challenging for student-athletes who are in the process of adjustment. In the current study, students mentioned the need to maintain a balance between the two roles of the student-athlete, and suggested that these competing roles, coupled with time demands, can interfere with academic and athletic performance. In addition, ISAs, especially those whose native language is not English, may struggle to meet the NCAA’s academic eligibility requirements (Connell, 2007). The ISA adjustment experience is complicated by the need to address the challenges of both international students and student-athletes. Japanese student-athletes must adjust to their roles as competitors to succeed at the high level of NCAA Division I competition. However, they must also adjust as students in a culturally and linguistically different environment. Regarding conditioning for competition, some participants felt that they could not eat as much as they would like because the foods available in the US were different from those in Japan. Previous studies have also noted that cafeteria food is often not appealing to ISAs (Rodriguez, 2014), and that it is difficult for ISAs to perform at a high level in the face of rapid dietary changes. In the current study, participants also reported that gaps in physique and athletic ability, tactical adjustments, and gaining playing opportunities were challenges.\n\nIn addition, participants reported that they faced challenges related to disadvantages in physical size and athletic ability when adapting competitively, making tactical adjustments, and gaining playing opportunities. However, these issues may be faced by all student-athletes, not just Japanese ISAs, because they depend not only on race and country of origin but also on the sport and the individual’s qualities and abilities as an athlete. In terms of personal emotional adjustment challenges, participants experienced homesickness, low motivation, and loneliness. Pierce et al. (2011) found that ISAs suffered from loneliness and homesickness after coming to the US, which is consistent with the current study’s findings. When non-native English speakers experience negative emotions such as sadness and suffering, their language skills may become impaired. Thus, individuals may struggle to acquire a new language unless they have sufficient language skills and experience in the country (Gass and Selinker, 2001).\n\nFurthermore, once an individual’s fear of not being able to succeed or perform concerning communication in a second language is experienced (Lin & Yi, 1997), the effects may persist for some time after they begin to become accustomed to their environment. Therefore, it can be inferred that for the participants in the current study, social anxiety caused by language and cultural adjustment difficulties may have been a significant factor in their emotional challenges, rather than simply being lonely while away from their parents. In addition, given that Gass and Selinker (2001) noted that social anxiety leads to avoidance of communication in society when speaking a second language, it can be assumed that the more significant this anxiety becomes, the more it affects the development of subsequent social relationships. Regarding the impact of cold and inclement weather on personal emotional adjustment, Searle and Ward (1990) found that religion, language, and climate are often the most important factors separating international students from their new cultural context, causing significant conflict. Popp (2007) also reported that the ISA recognizes climate adjustment as a challenge. Because Japan has both warm and cold climates in different regions, Japanese students studying in the US may find it difficult to adjust to the climatic differences between the region they are studying in and the region they are familiar with.\n\nRegarding the issue of school institutional adjustment, some participants expressed difficulty adapting to NCAA rules, which are very different from those of the school sports system in Japan. Although NCAA competitions and many of their rules are well-informed and well-known in the US, many ISAs are unfamiliar with the organization, its structure, and the stakeholders involved (Bale, 1991). Popp (2007) reported that better preparing international students for their departure to the US before leaving their home country can help to limit the impact of culture shock among ISAs. In addition, Popp (2007) recommends that students should be provided with information in advance, not only about athletics but also about college life in the US, student visas, and facts and figures about other international students’ college experiences. Adjustment challenges are commonly faced by ISAs who come from different cultural sporting backgrounds, and the current findings suggest that this also applies to Japanese ISAs.\n\nThis study involved several limitations. First, the study was limited to the perspectives of a small number of Japanese ISAs. Incorporating the voices of more participants could potentially improve the breadth of perspective and comprehensiveness of the findings. Second, this study did not allow for more extended conversations or interviews with participants. This may have limited the depth of information gathered and possibly participants’ openness to telling their stories. However, these limitations did not undermine the truthfulness of participants’ interview responses, their consent to share the recorded data, or the researcher’s interpretation through member checks or email follow-up. These limitations should be addressed in future studies.\n\nThe Japanese ISAs who participated in this study faced academic, social, athletic, personal-emotional, and institutional attachment challenges. In this context, Japanese ISAs differed from ISAs from other countries in several ways. Participants seemed to be perplexed by cultural differences, differences in communication styles, and systemic differences in academics and athletics between Japan and the US. First, as an academic adjustment issue, one respondent felt difficulty expressing her own opinions in discussions because of the gap between the lecture-oriented teaching style in Japan and her tendency to match her own opinions with the generally accepted correct answers. Therefore, Japanese students who wish to play in the ISA in the NCAA may find it valuable to gain experience through private services to familiarize themselves with the discussion-based teaching style or study abroad in a country where the teaching style is similar to that of the US. Alternatively, universities and coaches must recognize such issues and provide appropriate support. Second, regarding social adjustment challenges, participants struggled with cultural differences from Japan, such as differences in background experiences, perceptions, and communication styles.\n\nFurthermore, when building relationships with teammates and coaches, we found that some Japanese ISAs experienced communication-based prejudice because they were Asian minorities on the team. It would be difficult to deal with these perspectives, even for students living a typical university lifestyle in Japan. Therefore, to achieve smooth adjustment, it is necessary to build more relationships with people who are culturally and linguistically diverse, and, in the process, to deepen mutual understanding of various cultures and beliefs. In addition, universities and coaches need to understand the nature of Japanese ISAs and come to terms with each other. Third, differences in language and cultural backgrounds seemed to impact the challenges of competitive adjustment. Japanese student-athletes are not only required to adapt as competitors to succeed at the high level of NCAA Division I competition but also to adapt as students in a different culture and language environment at the same time. Therefore, Japanese ISAs need to understand the complex adjustments required, coupled with the linguistic and cultural distance between Japan and the US. Universities and coaches need to ascertain the extent to which they can adapt to Japanese ISAs, not only athletically but also in other aspects, and to provide appropriate support if there are factors affecting athletic performance because of insufficient adjustment. Fourth, participants felt homesickness, lack of motivation, and loneliness during the adjustment process. It was inferred that social anxiety resulting from language and cultural adjustment difficulties played significant roles in their emotional challenges, rather than simply experiencing loneliness as a result of being away from their parents. When Japanese ISAs are unable to adjust appropriately to their new environment, they tend to have adverse emotional reactions, which can be addressed by having positive dialogue and identifying the adjustment issues contributing to their reactions. Fifth, regarding institutional attachment issues, some participants seemed to have difficulty adapting to NCAA rules, which were very different from the school sports system in Japan. While the NCAA and many of its rules are well-informed and well-known in the US, many ISAs may not be familiar with the organization, structure, or member institutions. Therefore, to limit the impact of culture shock on Japanese ISAs, it is recommended that information be provided in advance about competition and college life in the US, student visas, and facts and figures about other international students’ college experiences. By identifying college adjustment challenges among Japanese ISAs, the current study encourages a better understanding of Japan, which is likely to become an increasingly important recruiting market for US universities in the future.",
"appendix": "Data availability\n\nAll datasets underlying the results cannot be sufficiently de-identified by redaction for uploading to a general data repository due to the sample size and nature of the conversations. Thus, the datasets are not available to share for ethical and privacy considerations as well as the guarantee given to the interviewees to maintain anonymity of the interview. A reader or reviewer may apply for access to the datasets by contacting the corresponding author (matsuo.hirokazu.ga@u.tsukuba.ac.jp) directly. The access will be available upon considering the request and permission received from the interviewees. The raw datasets are in Japanese.\n\nZenodo: Japanese international student-athletes’ adjustment experience at the National Collegiate Athletic Association. https://doi.org/10.5281/zenodo.7359143 (Matsuo et al., 2022).\n\nThis project contains the following extended data:\n\n• Interview guide in Japanese\n\nA summary of the interview questions in English can be found in Table 1.\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).\n\n\nAcknowledgments\n\nWe thank Benjamin Knight, MSc., from Edanz (https://jp.edanz.com/ac) for editing a draft of this manuscript. This study was conducted as a joint research project with second place Inc.\n\n\nReferences\n\nAllen K, Ingulsrud JE: Reading manga: Patterns of personal literacies among adolescents. Lang. Educ. 2005; 19(4): 265–280. Publisher Full Text\n\nAndrade M: International students and the first year of college. 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Soc. Inf. Sci. Technol. August 2006; 57(10): 1284–1295. Publisher Full Text\n\nMerriam SB: Qualitative Research and Case Study Applications in Education. Revised and Expanded from Case Study Research in Education. ERIC;1998.\n\nPalthe J: The relative importance of antecedents to cross-cultural adjustment: implications for managing a global workforce. Int. J. Intercult. Relat. February 2004; 28(1): 37–59. Publisher Full Text\n\nPatton MQ: Two decades of developments in qualitative inquiry: A personal, experiential perspective. Qualitative Social Work: Research and Practice. 2002; 1(3): 261–283. Publisher Full Text\n\nPierce DA, Popp N, Meadows B: Qualitative analysis of international student-athlete perspectives on recruitment and transitioning into american college sport. The Sport Journal. 2011; 14: 1–9.\n\nPopp N: International student -athlete perception of college sport and its effect on adjustment to college. 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Basingstoke, England:Palgrave Macmillan UK;2002nd edOctober 2002.\n\nStevenson H, Stigler JW: Learning gap: Why our schools are failing and what we can learn from Japanese and Chinese educ. Simon and Schuster;1992.\n\nTomika N, Paradis M: Cerebral processes involved in reading as a function of the structure of various writing systems. Neurosci. Res. 2002; 43(2): 87–89. PubMed Abstract | Publisher Full Text\n\nWells JC: Overcoming phonetic interference. English Phonetics. 2000; 3: 9–21.\n\nYin RK: Case study research: Design and methods. sage;2003; vol. 3. .\n\nZgonc E, et al.: Student-athlete ethnicity, 1999-2000-2009-2010. ncaa [r] student-athlete ethnicity report. National Collegiate Athletic Association (NJ1);2010."
}
|
[
{
"id": "158734",
"date": "11 Jan 2023",
"name": "Sarah Stokowski",
"expertise": [
"Reviewer Expertise Student-athlete experience",
"athlete 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\nThank you for the opportunity to review your paper. This paper had several grammatical errors that need to be corrected prior to publication. Although I feel this is an important topic, it has been studied. However, the authors are missing a lot of work here. Further, the studies that are cited are dated. I would suggested looking at the Journal of Issues in Intercollegiate Athletics (JIIA). I am also unsure why this study was limited to just athletes from Japan. More is needed to justify just Japanese athletes. The authors also say \"Asian\" students - but if this study is just Japanese athletes - why not just say Japanese athletes? There should also be a participant chart. Lastly, I've never seen a qualitative study with the exact same themes as another study. I have also never seen a qualitative study that uses Yin (who is a case study scholar) for interviews - the authors should consider using Patton. I would encourage the authors to come up with their unique codes.\nHere are the paper that should be added (the authors should also look at Kibaek Kim's work on intercollegiate sport assisting Asian students in adjusting to college campuses in the US).\nBeattie, M. A., & Turner, B. A. (2020). The impact of Person-Environment Fit on the academic satisfaction of Division II student-athletes. Journal of Issues in Intercollegiate Athletics, 13, 445-465.\nBoucher, M. E. (2017). Thrown to the wolves: The obstacles and barriers of international student athletes prior to and during enrollment in American universities (master’s thesis). Retrieved from BSU OneSearch. (3557)\nButler, B. N., Aicher, T. J., & Wieber, B. (2020). Junior college or the NCAA: The case of a US women’s tennis team with no US players. Journal of Global Sport Management, 5(1), 83–101. https://doi.org/10.1080/24704067.2019.1670089\nCharitonidi, I., & Kaburakis, A. (2022). The International Student-Athlete Experience: A Research Study Into the Transition Process of ISAs, From Their Home Country Into Life as an NCAA Student-Athlete. Sports Innovation Journal, 3(SI), 95-109. https://doi.org/10.18060/26060\nElliott, R., & Maguire, J. (2008). Thinking outside of the box: Exploring a conceptual synthesis for research in the area of athletic labor migration. Sociology of sport journal, 25(4), 482-497.\nFoo, C. E., Wells, J. E., & Walker, N. A. (2015). Do American college and university students endorse the recruitment of international student athletes? Journal of Contemporary Athletics, 9(3), 149-159.\nJara-Pazmino, E. S., Heere, B., Regan, T. H., Blake, C., & Southall, R. (2017). Coaches’ perception of organizational socialization process of international student-athletes and the effect of cultural distance: An exploratory study. International Journal of Exercise Science, 10(6), 875-899.\nLove, A., & Kim, S. (2011). Sport labor migration and collegiate sport in the United States: A typology of migrant athletes. Journal of Issues in Intercollegiate Athletics, 4, 90-104.\nManwell, A. K., Johnson, J. E., & Walker, K. B. (2021). International hispanic intercollegiate student-athletes in NCAA Division I: A qualitative exploration of culture in the student-athlete experience. Journal of Issues in Intercollegiate Athletics, 14, 524-546.\nNCAA. (2021b). International Student-Athletes. NCAA. https://www.ncaa.org/student-athletes/future/international-student-athletes\nNite, C. (2012). Challenges for supporting student-athlete development: Perspectives from an NCAA Division II athletic department. Journal of Issues in Intercollegiate Athletics, 5, 1–14.\nRidpath, B. D., Rudd, A., & Stokowski, S. (2020). Perceptions of European athletes that attend american colleges and universities for elite athletic development and higher education access. Journal of Global Sport Management, 5(1), 34–61. https://doi.org/10.1080/24704067.2019.1636402\nStokowski, S., Huffman, L. A., & Aicher, T. J. (2013). A comparative analysis of sport participation motivations of NCAA Division I student-athletes: An international focus. The Journal for the Study of Sports and Athletes in Education, 7(2), 131-148. https://doi.org/10.1179/1935739713Z.0000000009\nTurick, R., Feller, R., & Blom, L. (2020). Welcome to America! How can athletic departments better assist international student-athletes with their transition into the American university setting? Journal of Emerging Sport Studies, 4, 1-18.\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? Yes",
"responses": []
},
{
"id": "225174",
"date": "15 Dec 2023",
"name": "Guilherme Grubertt",
"expertise": [
"Reviewer Expertise Physical Education and Sport",
"Sport 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\nFirst of all, congratulations on your choice and on the time spent investing in this manuscript. However, there are still some gaps to be considered as publishable. Nevertheless, I added some contributions that may improve your research in the future. The main concept of this manuscript is very interesting and important. The study responds to the objective. Although, some points must be deepened. For example, justify in more detail the reason for evaluating only Japanese athletes. - Citations must be standardized in a single style; - It would be important to cite the main barriers faced by student-athletes according to each reference. Do not cite all references in a single parenthesis; - I suggest standardizing the identification of the sample in the paper (Japanese students Asian students or ISAs from Japan); - In addition to the references being cited repeatedly, they are not updated. There are several studies associated with the topic that could be cited; - To simplify comprehension, the contents of the subheadings \"sampling\" and \"participants\" can be presented in a table; - It is necessary to mention when (month and year) the data was collected; - There is a need for more characteristics of qualitative researchers with experience in the field and additional peers (e.g.professional background); - More information is needed on the continuous comparative analysis method; - To improve data analysis, I suggest using textual data analysis software (Iramuteq or MAXQDA); - It is necessary to revise the statement in the second line of the subtitle \"recommendations and conclusions\" because no comparison was conducted in the paper.\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? 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? Yes\n\nAre the conclusions drawn adequately supported by the results? Partly",
"responses": []
},
{
"id": "225176",
"date": "22 Dec 2023",
"name": "Robert Book Jr",
"expertise": [
"Reviewer Expertise Cultural sport 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\nI would like to thank the researchers for the time and efforts they spent in constructing the experiences on ISA’s transitioning from Japan to the USA. I accepted the opportunity to review this work because I am not only from the USA, but I have a lot of published research that focuses upon athletes who competed in the NCAA or transitioned to different countries and cultures. Therefore, this paper was an interesting (and I believe unique) topic that could add something to the literature. Overall, my impressions of the paper are a bit mixed and there needs to be significant changes, at least in my opinion.\nThe primary issue is that the references that the authors are relying upon are not only far too old and outdated, but you have also missed crucial areas of highly relevant research to this topic. One example would be that in the introduction you are inferring that athletic recruitment budgets are increasing over time, but citing Judson et al., (2004) hardly does anything to support your case as that is 20 years ago. In the introduction alone you attempt to support your case with references all the back in 1998, but that simply isn’t relevant research any longer. Of course, we are free to reference critical works that go back decades, but that is generally reserved to give credit to the founders of particular methodological or theoretical frameworks. In regards to relevant literature, it is apparent that the authors have missed a lot of relevant information that is crucial for framing this paper. Career pathway studies, dual career research, cultural transitions, and acculturation theory are all well documented areas of research and I urge the researchers to familiarize themselves in these areas. Researchers who specialize in this area include Natalia Stambulova, Noora Ronkinen, and Tatiana Ryba (Career pathways, cultural transitions, phases and transitions), Robert Schinke (Acculturation and cultural transitions), my own work is highly relevant regarding Black athletes in the NCAA, Kristoffer Henriksen (talent development), Paul Wylleman (career pathways), and William Massey and Meredith Whitley (NCAA athletes in universities). These are researchers who have a strong position within this field you are looking at and I think it is important to familiarize yourselves with their work and then link it to this study.\nI think the theoretical framing could be stronger using the cultural transition model Ryba et al. (20161), which is a highly regarded theoretical model used in top sport journals. You also should consider integrating acculturation theory (i.e., Schinke, Blodget, Middleton). The framework used here identified by Popp et al. was published in at least one journal where the authors have to pay to have it published, which does make me question the research quality.\nI would like to know more about the participants, primarily, were they in the US for the entirety of their studies? Were they actually playing on the top-level teams at a given university? One participant talked about being on a study abroad program, implying that they might have been in the US for a semester, which changes things dramatically as that is not the same as being enrolled at an American college or university for the entirety for 3-4 years.\nThe methods were ok, but no mention of the author’s philosophical positioning (i.e., epistomlogy and ontology)? This is an ever increasing demand in high quality qualitative research and I believe it is essentially a requirement at most international sport journals.\nContext. I think that more context should be given to the reader about the athletic career pathway in the United States. It is highly contextual and you can’t assume that the reader will understand. I have several papers that give a god overview of it.\nThe results were interesting in some places but I found a lot of the themes to be on the surface and would be expected. Also, only one of the themes directly pertained to sport, which seems little thin considering this is a sport journal. I believe that when most people read articles they want the authors to go deeper into ideas, concepts, and experiences that they haven’t deeply thought about, and I am not sure we see that developed fully here. For example, you mention that one athlete believed the scouting team coach seemed to ‘hate Asian’ people, and then it is never addressed again. Racism is a huge issue in the United States, and such tensions have roots firmly etched in WW2 and before. Forcing Japanese into internment camps, removing their legal rights and dignity, etc. Perhaps the participants felt no discrimination at all besides this one athlete, and if so, that is notable. Either way, these are the issues that must be expanded.\nI think the discussion is fine, but again is supported by outdated references which weaken the contribution. If the references are incorporated into the introduction as suggested, then these will inherently be integrated into the discussion as well.\n\nIs the work clearly and accurately presented and does it cite the current literature? No\n\nIs the study design appropriate and is the work technically sound? Partly\n\nAre sufficient details of methods and analysis provided to allow replication by others? Partly\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nNot applicable\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Partly",
"responses": []
}
] | 1
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https://f1000research.com/articles/11-1455
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https://f1000research.com/articles/11-1454/v1
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08 Dec 22
|
{
"type": "Brief Report",
"title": "COVID-19 among Indonesian ENT specialist and resident after second dose of Sinovac vaccination",
"authors": [
"Indra Zachreini",
"Jenny Bashiruddin",
"Susyana Tamin",
"Harim Priyono",
"Ika Dewi Mayangsari",
"Natasha Supartono",
"Anggina Diksita",
"Respati Wulansari Ranakusuma",
"Yussy Afriani Dewi",
"Sagung Rai Indrasari",
"Nyilo Purnami",
"Tengku Siti Hajar Haryuna",
"Juliandi Harahap",
"Eka Savitri",
"Tjandra Manukbua",
"Jenny Bashiruddin",
"Susyana Tamin",
"Harim Priyono",
"Ika Dewi Mayangsari",
"Natasha Supartono",
"Anggina Diksita",
"Respati Wulansari Ranakusuma",
"Yussy Afriani Dewi",
"Sagung Rai Indrasari",
"Nyilo Purnami",
"Tengku Siti Hajar Haryuna",
"Juliandi Harahap",
"Eka Savitri",
"Tjandra Manukbua"
],
"abstract": "Background: The purpose of this study was to obtain data on the prevalence of specialist doctors and Otorhinolaryngology-Head and Neck Surgery (ORL-HNS) residents in Indonesia who suffered from Coronavirus disease 2019 (COVID-19) after receiving the second dose of COVID-19 vaccination using the Sinovac vaccine (CoronaVac) and the length of time infected with COVID-19 after the second Sinovac vaccination. Methods: We performed a descriptive observational study, using a cross-sectional design. Data collection took place between August 2021 and October 2021. The respondents in this study included specialist doctors and ORL-HNS residents who worked in various hospitals in Indonesia who had received the second dose of the Sinovac vaccination. Data collection was performed by means of Self-reporting Online Survey Platform (Google Form). Results: This study included 1,530 respondents, and 54.2% of respondents were women. Respondents consisted of 68.6% ORL-HNS doctors and the rest were residents with an average age of 41.46 years old. The distance between the first and second doses of Sinovac was mostly under one month, which was 71.3% of the respondents. A total of 76.3% of respondents did not have co-morbid diseases. Based on this study, 16.9% of respondents suffered from COVID-19 after the second dose of Sinovac vaccination. The length of time suffering from COVID-19 after the second Sinovac vaccination was 3-6 months (9.7%). Conclusions: Based on this study, 16.9% of respondents suffered from COVID-19 after receiving the second Sinovac vaccination and 9.7% suffered from COVID-19 after 3-6 months of Sinovac vaccination.",
"keywords": [
"Covid 19",
"Sinovac vaccine",
"Vaccination"
],
"content": "Introduction\n\nCoronavirus disease 2019 (COVID-19) was first reported in Wuhan, Hubei Province, China, in December 2019. This disease is caused by the severe acute respiratory syndrome Coronavirus 2 (SARS-CoV-2).1 The World Health Organization (WHO) declared the COVID-19 outbreak a global health emergency on January 30, 2020, and a global pandemic on March 11, 2020.2,3\n\nEfforts to overcome COVID-19 were carried out, which included several strategies, one of which was vaccination. Vaccination is the most effective and efficient public health effort in preventing dangerous infectious diseases. Vaccines are a great hope as one of the primary weapons in controlling COVID-19.4\n\nIn the long run, COVID-19 vaccination aims to form herd immunity to protect all individuals and those unable to get vaccinated by reducing transmission of virus, and to lower morbidity and mortality for those affected (Ministry of Health Republic of Indonesia and National Guidelines on Vaccination Programme). This can only be achieved by high and even distribution of vaccination (WHO). Indonesia has carried out a national COVID-19 vaccination program since January 2021, which aims to create a condition of herd immunity (Ministry of Health Republic of Indonesia).\n\nWHO has recommended types of vaccines that have been evaluated and are safe to use, including the Pfizer, Moderna, AstraZeneca, Jessen, Sinopharm, and Sinovac vaccines (WHO Vaccine Recommendation). The types of vaccines are divided into several categories, namely DNA vaccines, RNA vaccines, non-replicating viral vector vaccines, inactivated vaccines, live attenuated vaccines, and vaccine subunits.4 Sinovac vaccine (CoronaVac) is one of the vaccines used in Indonesia according to the Decree of the Minister of Health of the Republic of Indonesia No. HK.01.07/Menkes/4638/2021 (Decree of the Minister of Health of the Republic of Indonesia).\n\nThe Sinovac vaccine has received approval for use in emergency conditions from the Food and Drug Supervisory Agency (Decree of the Minister of Health of the Republic of Indonesia). Although vaccination can prevent transmission of COVID-19, and it is possible to become infected after vaccination. Furthermore, the effectiveness of the Sinovac vaccine also decreases over time (Decree of the Minister of Health of the Republic of Indonesia). Based on this, we conducted a study on the effectiveness of the Sinovac vaccine on Otorhinolaryngology-Head and Neck Surgery (ORL-HNS) doctors and residents as seen from the presence of infection after the second dose of vaccination.\n\n\nMethods\n\nDue to the time sensitive and low risk nature of this project, we decided to carry out the research prior to getting ethical clearance as approval was taking longer than expected due to COVID-19 so we received retrospective approval for this study. Our original proposal included plans to start data collection in August 2021 following ethical approval and we planned to finish by the end of September 2021 and publish in October 2021, but due to the COVID-19 pandemic and part of our staff working remotely due to the Delta Variant of COVID-19, ethical clearance took longer to approve. Participant recruitment and distribution of questionnaires began on 1st September 2021, while data processing and finalizing the manuscript took place between February and March 2022. Ethical approval was granted by the Faculty of Medicine Universitas Indonesia in Dr. Cipto Mangunkusumo Hospital on 21st of February 2022 (approval number KET-188/UN2F1/ETIK/PPM.00.02/2022).\n\nInformed consent was acquired via written consent forms given to the participants as part of the questionnaire; the participant acknowledged that their data are to be used in a medical study.\n\nWe performed a descriptive observational study with a cross-sectional design conducted from September 2021 to March 2022. The inclusion criteria for this study were ORL-HNS doctors and residents who received the first and second Sinovac vaccinations and were willing to participate in this study.\n\nOutcome variables in this research included respondent demographic characteristic, the interval between first and second vaccination, comorbidity, and time between infection and the second dose of vaccination.\n\nFor this research, the data and measurement source were acquired directly from the respondent where they chose from list of choices given. The answers were then tabulated by frequency distribution (percentage).\n\nSince we acquired data through questionnaires, recall bias from the respondent might occur. To deal with this issue the respondents were encouraged to cross check the information with their vaccine certificate and PCR results they have within the time frame.\n\nWith a finite population of 1,657, the total number of respondents was 1,593 people consisting of ORL-HNS doctors and residents in all provinces in Indonesia. Inclusion criteria were vaccinated with Sinovac on first and second vaccination. Exclusion criteria were those who had not received their first and/or second dose vaccination. Minimum samples needed for this study were 322 samples, we used total sampling and included 1,530 samples once inclusion and exclusion criteria were applied.\n\nQuantitative variables in this study included age, time interval in vaccination time and time interval from second vaccination to infection.\n\nData collection was performed using a questionnaire by The Self-reporting Online Survey Platform (Google Form). The translated questionnaire is available as Extended data.5\n\nThe collected data were analyzed using IBM SPSS Statistics (RRID:SCR_016479) version 25.0 and evaluated using descriptive statistical methods and displayed in terms of frequency and percentage.\n\n\nResults\n\nTable 16 shows the number of respondents who took part in this survey, which included 1,530 respondents consisting of 68.6% ORL-HNS specialist doctors and 31.4% ORL-HNS residents. A total of 54.2% of respondents were women with the average age of respondents being 41.46 years old. Table 2 shows the time interval for the first and second Sinovac vaccination, which at most was under one month (71.3%). Table 3 shows that the most common type of comorbid disease was hypertension (47.7%). Based on Table 4, it was found that 16.9% of respondents were infected with COVID-19 after the second dose of Sinovac vaccination, and 7.4% of respondents were infected before vaccination. Based on the data presented in Table 5, the period from vaccination to COVID-19 infection was 3-6 months (57.3%) for specialists and residents who were infected after the second vaccination dose.\n\nCOVID-19, Coronavirus disease 2019.\n\n* At the time of data collection.\n\n\nDiscussion\n\nBased on this study, it was found that 258 (16.9%) of respondents suffered from COVID-19, after the second dose of vaccination. The length of time contracting COVID-19 after the second-highest dose of CoronaVac Sinovac vaccination was 3-6 months, as much as 9.7%.\n\nThe Sinovac vaccine produced by the Beijing-based pharmaceutical company Sinovac is a whole virus vaccine inactivated by adjuvant aluminum hydroxide (WHO). The efficacy of this vaccine based on phase 3 clinical trials conducted in Brazil was 51%, Turkey at 83.5%, and Indonesia at 65.3%. Furthermore, the efficacy of this vaccine remained the same in both groups with and without comorbidities, regardless of previous SARS-CoV-2 infection (WHO and Ministry of Health Republic of Indonesia). The effectiveness of Sinovac in preventing doctor visits and/or hospitalization was 74% (65–80%) in January to March 2021 in Indonesia, decreasing to 53% (33–67%). Meanwhile, the effectiveness in preventing death is 95% (53–99%) (National guidelines on vaccination programme).\n\nSinovac vaccine efficacy is indeed lower when compared to other types of vaccines, such as the Sinopharm vaccine showing 79% efficacy after two doses with an interval of 21 days (WHO, WHO interim, Ministry of Health Republic of Indonesia and Sinopharm Interim Recommendation); AstraZeneca vaccine has 72% efficacy after two standard doses with intervals varying from four weeks to 12 weeks (WHO interim); Moderna vaccine has 94% efficacy after receiving two doses (WHO interim), Pfizer vaccine has 95% efficacy after receiving the second dose with an interval of three weeks between the first and second doses of vaccine (WHO interim), the Sputnik V vaccine had an efficacy of 91.6% after receiving the second dose, and the Novavax vaccine had an efficacy of 89.7%.7,8\n\nThese results differ from a study conducted by Menni (2021), who found that the risk of being infected with COVID-19 after vaccination was more significant in individuals above the age of 55 years than those under 55 years old. This research also found that respondents with at least one comorbidity had a greater risk of being infected with COVID-19 after vaccination.9 Based on a report by WHO, COVID-19 vaccination with the Sinovac vaccine has been proven in clinical trial participants with co-morbid obesity and hypertension (WHO).\n\n\nConclusions\n\nBased on the present study, 16.9% of respondents suffered from COVID-19 after receiving the second Sinovac vaccination and 9.7% suffered from COVID-19 three to six months after the Sinovac vaccination.",
"appendix": "Data availability\n\nFigshare: MASTER DATA of Covid-19 among Indonesian ENT Specialist and Resident after Second Dose of Sinovac Vaccination.xlsx. https://doi.org/10.6084/m9.figshare.21173686. 6\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).\n\nFigshare: Questionnaire Covid-19 among Indonesian ENT Specialist and Resident after Second dose of Sinovac Vaccination. https://doi.org/10.6084/m9.figshare.21229742. 5\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\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\nBurki TK: Coronavirus in China. Lancet Respir. Med. 2020 Mar; 8(3): 238. PubMed Abstract | Publisher Full Text | Free Full Text\n\nHan X, Xu P, Ye Q: Analysis of COVID-19 vaccines: Types, thoughts, and application. J. Clin. Lab. Anal. 2021; 35(9): e23937. PubMed Abstract | Publisher Full Text | Free Full Text\n\nAditama TY: Covid-19 dalam Tulisan Prof. Tjandra. Jakarta:Health Research and Development Publishing Institute;2020; 70–80.\n\nZachreini I, et al.: Questionnaire COVID-19 among Indonesian ENT Specialist and Resident after Second Dose of Sinovac Vaccination.docx. figshare. [Dataset]. 2022. Publisher Full Text\n\nZachreini I, et al.: MASTER DATA of Covid-19 among Indonesian ENT Specialist and Resident after Second Dose of Sinovac Vaccination.xlsx. figshare. [Dataset]. 2022. Publisher Full Text\n\nLogunov D: Safety and efficacy of an rAd26 and rAd5 vector-based heterologous prime-boost COVID-19 vaccine: an interim analysis of a randomized controlled phase 3 trial in Russia. J. Lancet. 2021; 397(10275): 671–681. PubMed Abstract | Publisher Full Text | Free Full Text\n\nHeath P, et al.: Safety and Efficacy of NVX-CoV2373 Covid-19 Vaccine. N. Eng. J. Med. 2021; 385(1): 1172–1183.\n\nMenni, et al.: Vaccine side-effects and SARS-CoV-2 infection after vaccination in users of the COVID Symptom Study app in the UK: a prospective observational study. Lancet Infect. Dis. 2021; 21(7): 939–949. PubMed Abstract | Publisher Full Text | Free Full Text"
}
|
[
{
"id": "198125",
"date": "04 Sep 2023",
"name": "Rivaldo Steven Heriyanto",
"expertise": [
"Reviewer Expertise Pediatrics",
"General Medicine",
"Internal Medicine",
"Public Health"
],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nOverall: A well written report that provided a good insight on the specific matters that are studied.\nIntroduction: the introduction is good, it clearly and accurately presented the work and recent issue. However, I would like to know what are the author's reason for specifically selecting ENT specialist and residents as the subject of the study? In the last paragraph of the introduction, the author suddenly explained that the study will be conducted on ENT specialist and resident without explaining the reason of selection prior to it\nMethod: The study design is appropriate for this study, however, if we are talking about the details on the method and analysis, I still find it lacking. It would be better if the author could elaborate more on the method and analysis. For example, has the translated questionnaire been validated before? If yes, by what method? What other bias might occurred and how the authors mitigate that bias?\nResult: The result are well done, I don't have any comments.\nDiscussion: The discussion is sufficient, I have no further comment.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Partly\n\nIf applicable, is the statistical analysis and its interpretation appropriate? Yes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes",
"responses": []
},
{
"id": "228001",
"date": "18 Dec 2023",
"name": "Nurul Hayati Mohamad Zainal",
"expertise": [
"Reviewer Expertise 1. Human Physiology2. Proteomics3. Paediatrics and Maternal Reproductive Health4. Allergy and Immunology5. Molecular Biology6. Medical 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\nF1000 Manuscript’s Feedback Review Comments to the Author Interesting manuscript. Good work. I have a few feedback. For overall feedback:\nKindly ensure the references are properly cited throughout the manuscript. It would be interesting to know the different percentages of COVID-19 infection in terms of different types of vaccine used.\n\nTitle\n\nSuggest adding “COVID-19 Infection…” and the time duration (e.g. during the pandemic or post-second wave).\nAbstract\nWritten clearly.\nIntroduction\nSuggest\nAdding a bit of info on other types of vaccines and the prevalence of each vaccine used in Indonesia. Is Sinovac the only vaccine used? Explaining the recommended time interval between the first and second Sinovac vaccination.\n\nMethods\nStudy design\n\nKindly state clearly the inclusion and exclusion criteria of the study population in one paragraph. It would be interesting to know if there are other findings when other statistical analyses are used (e.g. associations with the independent variables using either the Pearson chi-square or Fisher’s exact test).\n\nResults\nWritten clearly.\nDiscussion\nWritten clearly.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate? Yes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes",
"responses": []
}
] | 1
|
https://f1000research.com/articles/11-1454
|
https://f1000research.com/articles/11-1451/v1
|
08 Dec 22
|
{
"type": "Case Report",
"title": "Case Report: An exceptionally large spindle cell lipoma of the popliteal fossa",
"authors": [
"Rafik Elafram",
"Majdi Ben Romdhane",
"Nayssem Khessairi",
"Sarra Ben Rjeb",
"Saif Toumi",
"Majdi Sghaier",
"Majdi Ben Romdhane",
"Nayssem Khessairi",
"Sarra Ben Rjeb",
"Saif Toumi",
"Majdi Sghaier"
],
"abstract": "Background: Spindle cell lipoma (SCL) is an uncommon subtype of lipomas. It usually occurs in the posterior side of the trunk such as neck, back and shoulders and rarely touches the extremities. Only four cases of SCL of the knee and no cases of SCL of the popliteal fossa have been reported in the literature. We present herein the largest case of SCL of the popliteal fossa and knee to our knowledge. Case presentation: A 75-year-old woman presented with a slow growing swelling of the left knee. Physical examination showed a 12 cm well-limited painless mass of the left popliteal fossa. The mass was pediculated with a central ulceration. MRI scan showed a tumoral mass of the left popliteal fossa with some non-dilated vessels draining to the long saphenous vein. The mass had no evidence of invasion of the underlying tissues. The patient underwent a surgical resection of the mass with postoperative recovery. The histological examination revealed a tumoral proliferation diffusely expressing CD34 without malignancy evidence, and a Ki67 proliferation index <1%. This morphological and immunohistochemical aspect is typical of a SCL. Clinical discussion: SCL is a rare histological variant of lipoma occurring mostly in the posterior side of the upper trunk. SCL of the knee or the popliteal fossa are exceptional. Clinically, it presents as a gradually increasing painless mass with a long evolving history. Histologically, these tumors are made of spindle-shaped cells, rope-like collagen fibers and mature adipocytes surrounded by a fibrous capsule. Immunohistologically, spindle cell lipomas usually show diffuse and strong expression of CD34, and to a lesser degree vimentin. The optimal treatment of SCL is a surgical marginal resection. Conclusions: SCL represents an infrequent subtype, and its atypical presentation should push clinicians to further investigations in order to rule out more worrisome malignancies.",
"keywords": [
"lipoma",
"spindle cells",
"spindle cell lipoma",
"popliteal fossa",
"case report"
],
"content": "Introduction\n\nSpindle cell lipoma (SCL) is an infrequent benign tumor of the subcutaneous fat tissue. It usually touches the shawl region, but rarely the scalp, the face, or the extremities.1 SCL of the knee represents less than 1% of all spindle cell lipomas; to date only a few cases are reported in the literature.1–3 No cases of SCL of the popliteal fossa have been reported. The classical reported tumors have mostly been between 1 and 5cm in size which are significantly smaller than the one presented in this case. We present herein the first and largest spindle cell lipoma of the popliteal fossa to our knowledge. This case report has been reported in line with the criteria.\n\n\nCase presentation\n\nA 75-year-old woman, with a medical history of hypertension, presented with a swelling of the left knee evolving for four years since November 2017. The mass was initially sub-centimetric and gradually increased in size leading the patient to seek medical attention. Physical examination showed a 12 cm firm, well-limited, and painless mass of the left popliteal fossa (Figure 1). This mass was mobile, pediculated with a central ulceration leaking pus. An ultrasound scan of the left knee was performed showing a well-limited, non-vascularized skin mass of 10 cm, with no popliteal cysts or vascular lesions. MRI scan of the left leg revealed a tumor mass in the popliteal fossa measuring 98 x 55 x 8 mm with some non-dilated vessels draining to the long saphenous vein. There was no infiltration of the underlying tissues (Figure 2). The patient underwent a surgical total resection of the mass. Postoperative care was uncomplicated. The intervention was performed by a senior orthopedic surgeon in the Orthopedic Surgery Department of the Internal Security Forces Hospital, La Marsa, Tunisia (Figure 3). Gross examination showed a mass of 11 x 9 x 6 cm with a cutaneous ulceration of 3 x 4 cm. There was no evidence of macroscopic extension to the underlying subcutaneous tissue. Histological examination showed a benign mesenchymal proliferation associating foci of well-differentiated and mature adipocytes and myxoid and collagenous patches of variable thickness. These patches were composed of spindle-shaped cells with elongated nuclei without any atypical character or mitosis figures, mixed with collagenous clusters. Numerous mast cells were also seen. No vascular thrombi, lipoblasts, or necrosis were seen. There were no signs of malignancy. On immunohistochemical study, this tumor proliferation diffusely expressed CD34. Desmin expression was negative and the Ki67 proliferation index was <1% (Figures 4–6) This morphological and immunophenotypic appearance was compatible with a spindle cell lipoma. The resection was total and there was no tumor residue. In July 2022, after one year of follow-up, the patient had no signs of local recurrence. She was able to do all her daily activities without any sensory-motor disorder.\n\n\nDiscussion\n\nSCL is an uncommon histological variant of lipoma (representing 1.5% of all lipomas) first described by Enzinger and Harvey in 1975.4,5 It is a benign tumor of the subcutaneous fat tissue that occurs mostly in the posterior side of the upper trunk such as the neck, the back, and the shoulders. Atypical locations are possible, it may touch the face, the scalp, the upper and lower extremities, the retroperitoneum, but rarely the knee or the popliteal fossa.6–10 Only four cases of SCL of the knee have been reported, these cases are summarized in Table 1. SCL has a noteworthy predilection for men with a percentage of 90% occurring usually in the second and third decades.4 Clinically, the spindle cell lipoma presents as a gradually increasing painless mass with a long history (more than one year in 75% of cases). The tumor may reach a considerable size leading the patient to seek medical attention. Spindle cell lipomas usually have clear boundaries, are firm, mobile and without associated cutaneous lesions, ulcerations are rarely noted.7,11 SCL usually measures between 1 and 5 cm in greatest dimension, rarely exceeding 10 cm. The rare cases with extraordinary size or atypical anatomical location require further investigations to rule out more worrisome malignancies.9,12–15\n\nRadiologically, the images of the SCL are variable and no pathognomonic signs are identified. In most cases, tumor images are composed of a relatively equal ratio of spindle cells and fatty tissue. This ratio is variable, and the aspect may overlap that of liposarcoma or hibernoma. Intense enhancement of the non-adipose component supports the diagnosis of spindle cell lipoma.2,16 Histologically, SCL have different textures, from soft to tough, usually without necrosis or bleeding. Most of the tumors are made of spindle-shaped cells, rope-like collagen fibers and mature adipocytes surrounded by a fibrous capsule. Mast cells are numerous in a significant number of cases, small aggregates of lymphocytes are seen only occasionally. Different amounts of adipocytic component are present and may be accompanied by a myxoid or collagenous matrix. Therefore, multiple subtypes of spindle cell lipoma may exist such as fibrous, myxoid, fat-rich, low-fat, and pseudo angiomatous type.1,17 Most cells do not have atypia or high mitotic activity. Immunohistologically, spindle cell lipomas usually show diffuse and strong expression of CD34, and to a lesser degree vimentin. Proliferation index Ki-67 is <3% in 99% of cases. Due to the diversity of histological morphotypes of SCL, it is sometimes diagnosed as other benign or malignant tumors such as solitary fibrous tumor and shuttle cell liposarcoma, therefore immunohistochemical staining combined with molecular pathology examination can confirm the diagnosis. Benign spindle cell lesions are usually underrecognized and can represent potential pitfalls of malignancy, particularly in a small biopsy. The main differential diagnoses of spindle cell lipoma are elastrofibroma, solitary fibrous tumors, angiomyolipoma, myxoid liposarcoma (for the myxoid type of SCL) and mammary myofibroblastoma.16,18 Spindle cell lipoma and pleomorphic lipoma share many known non-pathognomonic genetic chromosomal abnormalities specifically losses at 16q and 13q chromosomes, but these abnormalities are not constant and do not interfere with the diagnosis.12,19 The standard treatment of SCL is the surgical marginal resection, post excision recurrence rate is stated to be <1% with adequate marginal resection.3,9\n\n\nConclusions\n\nIt was noted that SCL is similar to many benign and malignant soft tissue tumors in pathologic morphology. Therefore, pathologists and clinicians should be conscious of multiple variant subtypes of SCL when encountering spindle cell-associated soft tissue tumors to avoid misdiagnosis.\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\nChen S, Huang H, He S, et al.: Spindle cell lipoma: clinicopathologic characterization of 40 cases. Int. J. Clin. Exp. Pathol. 2019; 12(7): 2613–2621.\n\nJelinek JS, Wu A, Wallace M, et al.: Imaging of spindle cell lipoma. Clin. Radiol. 2020 May; 75(5): 396.e15–396.e21. Publisher Full Text\n\nGoto T, Motoi N, Motoi T, et al.: Spindle cell lipoma of the knee: a case report. J. Orthop. Sci. Off. J. Jpn. Orthop. Assoc. 2004; 9(1): 86–89. Publisher Full Text\n\nEnzinger FM, Harvey DA: Spindle cell lipoma. Cancer 1975 Nov; 36(5): 1852–1859. Publisher Full Text\n\nKallen ME, Hornick JL: The 2020 WHO Classification: What’s New in Soft Tissue Tumor Pathology? Am. J. Surg. Pathol. 2021 Jan 6; 45(1): e1–e23. Publisher Full Text\n\nUd Din N, Zhang P, Sukov WR, et al.: Spindle Cell Lipomas Arising at Atypical Locations. Am. J. Clin. Pathol. 2016 Oct; 146(4): 487–495. PubMed Abstract | Publisher Full Text\n\nKo JS, Daniels B, Emanuel PO, et al.: Spindle Cell Lipomas in Women: A Report of 53 Cases. Am. J. Surg. Pathol. 2017 Sep; 41(9): 1267–1274. Publisher Full Text\n\nLiang Z, Zang Y, Jing Z, et al.: Hypopharyngeal spindle cell lipoma: A case report and review of literature. Medicine (Baltimore) 2021 May 7; 100(18): e25782. Publisher Full Text\n\nAkaike K, Suehara Y, Takagi T, et al.: Spindle cell lipoma of the wrist, occurring in a distinctly rare location: a case report with review of literature. Int. J. Clin. Exp. Pathol. 2015; 8(3): 3299–3303.\n\nAzar SS, Buen F, Chia JJ, et al.: Spindle Cell Lipoma Arising from the Supraglottis: A Case Report and Review of the Literature. Head Neck Pathol. 2021 Dec; 15(4): 1299–1302. Publisher Full Text\n\nChandrashekhar P, Jose M, Dadhich M, et al.: Spindle cell lipoma: a case report and review of literature. Kathmandu Univ Med J KUMJ. 2012 Jun; 10(38): 92–95. PubMed Abstract | Publisher Full Text\n\nJohn M, Patel S, Joseph G: Exceptionally large, atypically located spindle cell lipoma. BMJ Case Rep. 2019 Oct 31; 12(10): e232209. PubMed Abstract | Publisher Full Text\n\nLincoln M, Royer M: Uncommon Tumor, Uncommon Location: A Dermal-Based Spindle Cell/Pleomorphic Lipoma. Am. J. Dermatopathol. 2016 Aug; 38(8): e122–e124.\n\nBahadır B, Behzatoğlu K, Hacıhasanoğlu E, et al.: Atypical spindle cell/pleomorphic lipomatous tumor: A clinicopathologic, immunohistochemical, and molecular study of 20 cases. Pathol. Int. 2018 Oct; 68(10): 550–556. PubMed Abstract | Publisher Full Text\n\nMariño-Enriquez A, Nascimento AF, Ligon AH, et al.: Atypical Spindle Cell Lipomatous Tumor: Clinicopathologic Characterization of 232 Cases Demonstrating a Morphologic Spectrum. Am. J. Surg. Pathol. 2017 Feb; 41(2): 234–244. Publisher Full Text\n\nBancroft LW, Kransdorf MJ, Peterson JJ, et al.: Benign fatty tumors: classification, clinical course, imaging appearance, and treatment. Skelet. Radiol. 2006 Oct; 35(10): 719–733. Publisher Full Text\n\nBillings SD, Folpe AL: Diagnostically challenging spindle cell lipomas: a report of 34 “low-fat” and “fat-free” variants. Am. J. Dermatopathol. 2007 Oct; 29(5): 437–442. PubMed Abstract | Publisher Full Text\n\nMagro G: Differential Diagnosis of Benign Spindle Cell Lesions. Surg. Pathol. Clin. 2018 Mar; 11(1): 91–121. Publisher Full Text\n\nPanagopoulos I, Gorunova L, Lund-Iversen M, et al.: Cytogenetics of Spindle Cell/Pleomorphic Lipomas: Karyotyping and FISH Analysis of 31 Tumors. Cancer Genomics Proteomics. 2018 Jun; 15(3): 193–200. PubMed Abstract\n\nRafik E, Nayssem K, Majdi Ben R, et al.: The lipoma-like hibernoma: A case report of a rare entity. Radiology Case Reports. 2023; 18(1): 75–78."
}
|
[
{
"id": "192066",
"date": "14 Aug 2023",
"name": "Pooja K. Suresh",
"expertise": [
"Reviewer Expertise Surgical pathology",
"Gastrointestinal Pathology",
"Head and neck Pathology",
"Bone and soft tissue pathology",
"Genitourinary pathology with immunohistochemistry."
],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nWell-written case report. These are a few of my comments.\nThe radiological features of SCL can be described in detail and the points to differentiate it from its differentials can also be added.\n\n\"Therefore, multiple subtypes of spindle cell lipoma may exist such as fibrous, myxoid, fat-rich, low-fat, and pseudo angiomatous type.\" The different histological variants can be described.\n\nThe definite morphological features that can help to differentiate SCL from atypical lipomatous tumor/ well differentiated liposarcoma and other differentials (elastrofibroma, solitary fibrous tumors, angiomyolipoma, myxoid liposarcoma (for the myxoid type of SCL) and mammary myofibroblastoma) can be added.\n\nIs the background of the case’s history and progression described in sufficient detail? Yes\n\nAre enough details provided of any physical examination and diagnostic tests, treatment given and outcomes? Yes\n\nIs sufficient discussion included of the importance of the findings and their relevance to future understanding of disease processes, diagnosis or treatment? Partly\n\nIs the case presented with sufficient detail to be useful for other practitioners? Yes",
"responses": []
},
{
"id": "213525",
"date": "30 Oct 2023",
"name": "Xingpei Hao",
"expertise": [
"Reviewer Expertise Pathology",
"cancers",
"acute and chronic infectious diseases."
],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe manuscript described a single rare case report of spindle cell lipoma. It is interesting and deserves for consideration. However, the manuscript cannot be accepted in the current version before addressing some issues bellow.\nThe abstract needs to be written more concise and logical. The case presentation and clinical discussion needs to be merged together to avoid repeated sentences.\n\nCD34 is not a specific marker for SCL. Rb1 and MDM2 should be immunostained.\n\n“This case report has been reported in line with the criteria” needs to be specified for what criteria followed.\n\n“The intervention was performed by a senior orthopedic surgeon in the Orthopedic Surgery Department of the Internal Security Forces Hospital, La Marsa, Tunisia” is redundant.\n\nThis reference1 should be cited to show SCL occurring in the extremity.\n\nThe English writing needs to be further improved to avoid any grammar or wording issues. For example,\" We present herein the largest case of SCL of the popliteal fossa and knee to our knowledge\". It should be “We present herein the largest SCL of the popliteal fossa and knee to our knowledge”.\n\nThe conclusion at the end also needs to show the most important findings in this patient.\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": []
}
] | 1
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https://f1000research.com/articles/11-1451
|
https://f1000research.com/articles/11-1450/v1
|
07 Dec 22
|
{
"type": "Research Article",
"title": "Evaluation of cytosine conversion methods for whole-genome DNA methylation profiling",
"authors": [
"Shyaron Poudel",
"Brett Hale",
"Asela J. Wijeratne",
"Shyaron Poudel",
"Brett Hale"
],
"abstract": "Background: DNA methylation, the most common epigenetic modification, is defined as the removal or addition of methyl groups to cytosine bases. Studying DNA methylation provides insight into the regulation of gene expression, transposon mobility, genomic stability, and genomic imprinting. Whole-genome DNA methylation profiling (WGDM) is a powerful tool to find DNA methylation. This technique combines standard whole-genome sequencing methodology (e.g., Illumina high-throughput sequencing) with additional steps where unmethylated cytosine is converted to uracil. However, factors such as low cytosine conversion efficiency and inadequate DNA recovery during sample preparation oftentimes render poor-quality data. It is therefore imperative to benchmark sample preparation protocols to increase sequencing data quality and reduce false positives in methylation detection. Methods: A survey analysis was performed to investigate the efficiency of the following commercially available cytosine conversion kits when coupled with the NEBNext® Ultra™ DNA Library Prep Kit for Illumina (NEB): Zymo Research EZ DNA Methylation™ kit (hereafter known as Zymo Conversion kit), QIAGEN EpiTect Bisulfite kit (hereafter known as QIAGEN Conversion kit), and NEBNext® Enzymatic Methyl-seq Conversion Module (hereafter known as NEB EM-seq kit). Input DNA was derived from soybean (Glycine max [L.] Merrill) leaf tissue. Results: Of those tested, the QIAGEN Conversion kit provided the best sample recovery and the highest number of sequencing reads, whereas the Zymo Conversion kit had the best cytosine conversion efficiency and the least duplication. The sequence library obtained with the NEB EM-seq kit had the highest mapping efficiency (percentage of reads mapped to the genome). The data quality (defined by Phred score) and methylated cytosine call were similar between kits. Conclusions: This study offers the groundwork for selecting an effective DNA methylation detection kit for crop genome research.",
"keywords": [
"DNA methylation",
"Methylation profiling",
"Whole-Genome Bisulfite Sequencing (WGBS)",
"Enzymatic methyl-seq (EM-seq)",
"Bisulfite",
"Cytosine",
"Uracil"
],
"content": "Introduction\n\nDNA methylation is a key epigenetic regulator of many biological processes, including transposon activation, heterosis, biotic and abiotic stress response, development, and reproduction (Moore et al., 2013). Whole-genome DNA methylation profiling (WGDM) is among the most efficient techniques available for the detection of DNA methylation, providing >90% single-nucleotide-based genome-wide coverage for cytosines followed by guanine (CpG) residues (Chatterjee et al., 2017). WGDM is similar to the traditional whole-genome sequencing technique with additional steps of cytosine conversion to uracil (Plongthongkum et al., 2014; Li et al., 2018). Of deployed cytosine conversion techniques, sodium bisulfite (SB) conversion is a gold standard process that utilizes single-base-level chemical treatment to convert cytosine to uracil while leaving 5-methylcytosine (5-mC) unaffected prior to PCR enrichment (Figure 1). During PCR amplification, uracil is converted to thiamine, allowing researchers to perform high-throughput sequencing to determine the average methylation level in a sample (Feng et al., 2020). Although considered a gold standard, several limitations can arise through SB treatment, such as low conversion efficiency (failed conversion of non-methylated cytosines to uracil) and DNA degradation. These can impair the efficiency of downstream steps such as PCR amplification, library yield, and false detection of methylated cytosine (Hernández et al., 2013; Iurlaro et al., 2016; Tierling et al., 2018).\n\nCreated with BioRender.com. WGBS, whole-genome bisulfite sequencing; EM-seq, enzymatic methylation sequencing; APOBEC3A, apolipoprotein B mRNA editing enzyme catalytic polypeptide-like 3A.\n\nAn approach known as enzymatic methyl-seq (EM) conversion has recently been developed to circumvent the limitations of SB conversion (Vaisvila et al., 2021). In the first step of this method, the 5-mC is converted to 5-hydroxymethylcytosine (5-hmC), then 5-formylcytosine (5-fC), and eventually to 5-carboxylcytosine (5-caC) using the ten-eleven translocation (TET) enzyme, which protects the methylated cytosines to be converted in downstream steps (Ito et al., 2011). In the second reaction, an enzyme known as apolipoprotein B mRNA editing enzyme catalytic polypeptide-like 3A (APOBEC3A) deaminates non-methylated cytosines into uracil (Figure 1) (Wijesinghe and Bhagwat, 2012; Schutsky et al., 2017). The final sequences containing methylated cytosines remain cytosines, while non-methylated cytosines appear as thymine (like in SB conversion-mediated methylation detection). Therefore, the same analytical methods can be used for both EM- and SB-converted DNA (Feng et al., 2020).\n\nRecent data indicate EM conversion provides superior results compared to SB treatment. In a current research, Feng et al., (2020) investigated DNA methylation in Arabidopsis thaliana using the enzymatic methylation sequencing (EM-seq) method and compared their findings to whole-genome bisulfite sequencing (WGBS) data. They reported EM-seq provided better data than that of WGBS. Specifically, libraries prepared with EM-seq kits had higher mapping and lower duplication rates, lower background noise, higher average coverage, and higher total cytosine coverage between replicates (Feng et al., 2020). In another study using human gDNA, similar results were obtained where EM-seq libraries outscored bisulfite-converted libraries in most of the examined parameters, including coverage, duplication, sensitivity, and nucleotide composition (Vaisvila et al., 2021). Additionally, even with extremely low levels of DNA (100 pg), EM-seq proved to be a more reliable method for detecting methylation state than WBGS, which requires 10–200 ng DNA input (Vaisvila et al., 2021).\n\nHowever, optimized sample preparation protocols are only available for a handful of species and are missing for many important crop plants. In addition, most existing benchmark methods have been performed for mammalian genomes, which are different from plant genomes (e.g., plant genomes are enriched with repetitive elements, as well as CHG and CHH methylation). Therefore, in this study, we have compared two commercial SB conversion kits: Zymo Conversion kit and QIAGEN Conversion kit with an EM conversion kit, NEB EM-seq kit, using soybean (Glycine max [L.] Merrill) DNA samples. Further, we have benchmarked the use of these three kits with the NEBNext® Ultra™ DNA Library Prep Kit for Illumina (NEB) for sample preparation for high-throughput sequencing.\n\n\nMethods\n\nSeeds of soybean genotype Williams 82 were sown in a Miracle-Gro Moisture Control potting medium and grown at 25°C, 16 h d−1 light at 230 to 365 μM m−2 s−1, and 90% relative humidity in a growth chamber. At the V2 developmental stage (Fehr and Caviness, 1977), a trifoliate leaf was collected for DNA isolation (Figure 2).\n\nCreated with BioRender.com. WGBS, whole-genome bisulfite sequencing; EM-seq, enzymatic methylation sequencing; NEB, New England BioLabs.\n\nA detailed protocol can be found on protocols.io. Genomic DNA (gDNA) was isolated using the Zymo Research Quick-DNA Plant/Seed Miniprep kit (Cat #D6020; Irvine, CA, USA). DNA concentration was measured using a Qubit fluorometer (Thermo Fisher Scientific; Waltham, MA, USA) coupled with a Thermo Fisher Scientific dsDNA High Sensitivity Assay kit (Cat #Q32851). DNA purity was assessed from 260/230 and 260/280 nm absorbance ratios using a NanoDrop ND-1000 spectrophotometer (Thermo Fisher Scientific). Furthermore, the quality and size of obtained DNA was determined using gel electrophoresis (Fisher Scientific gel casting and electrophoresis apparatus (Cat #FB-SB-1316); 1% agarose gel with 1X Tris acetate-EDTA buffer (ThermoFisher; Cat # B49). Nucleic Acid was stained with Biotium GelRed Nucleic Acid Gel Stain (Fisher Scientific; Cat # NC9594719) and gel imaging was performed using LiCOR ODYSSEY imager (Serial # OFC1321) under a 520 nm channel. As a non-methylated control, the soybean gDNA was spiked with Escherichia coli gDNA (Zymo Research, Cat #D5016) representing 1% of the total gDNA input. The spiked sample was then separated into three aliquots (each 65 μL), which were sheared using Qsonica sonicators (Newtown, CT, USA). Sonication settings entailed 15 sec on/90 sec off for 8 cycles at a 20 kHz frequency, resulting in DNA fragments of approximately 350 bp. Sheared aliquots were used for library preparation, as described below.\n\nSequencing libraries were prepared using the NEBNext® Ultra™ II DNA Library Prep Kit for Illumina® (New England Biolabs; Cat #E7645S; Ipswich, MA, USA). A total of 200 ng (for NEB EM-seq kit) or 1 μg (Zymo and QIAGEN Conversion kits) of starting DNA was used based on the manufacturer’s instructions (Figure 2). The sheared DNA fragments were repaired, and Illumina Methylated Adaptors were ligated using NEBNext® Multiplex Oligos for Illumina (NEB Cat #E7535S/L). Adapter-ligated fragments were then purified using Solid Phase Reversible Immobilization (SPRI) magnetic beads (Beckman Coulter Inc.; Cat #B23317; Brea, CA, USA). For the cytosine conversion, the adapter-ligated, purified DNA samples were processed with either a Zymo Conversion kit (EZ DNA Methylation™ Kit, Zymo Research; Cat #D5001), QIAGEN Conversion kit (EpiTect Bisulfite Kit, QIAGEN; Cat #59104), or NEB EM-seq kit (NEBNext® Enzymatic Methyl-seq Conversion Module, New England Biolab; Cat #E7125S/L) (Figure 2).\n\nFor SB conversion using the Zymo Conversion kit, DNA was incubated with CT Conversion Reagent (Zymo Research; Cat #D5001-1) (Table 3 and Figure 1) in a thermocycler for approximately 16 hrs. The converted DNA samples were then desulphonated using M-Desulphonation Buffer (Zymo Research; Cat #D5001-5). For SB conversion using the QIAGEN Conversion kit, the sample was incubated in a thermocycler for approximately 5 hrs with the Bisulfite mix (Table 2 and Figure 1) and the DNA protection buffer provided in the kit. Converted DNA samples were then treated with Buffer BD from the kit for desulphonation. For EM conversion, the sample was first treated with TET2 (NEB Cat #E7130AVIAL; part of the NEBNext® Enzymatic Methyl-seq Conversion Module) and APOBEC (NEB Cat #E7133AVIAL) (Table 1 and Figure 1) enzymes in a series of steps, which ultimately converted cytosines to uracil, allowing for the detection of 5 mC and 5 hMC.\n\nQuality was determined by library recovery (%), number of unprocessed and preprocessed raw sequences, average length of preprocessed sequences (bp), duplication rate (%), GC content (%), mapping efficiency (%), total coverage (X), and cytosine conversion efficiency (%). NEB, New England BioLabs.\n\nConverted samples were purified using Beckman Coulter SPRIselect magnetic beads (Cat #B23317). The barcoding of converted DNA fragments was carried out with NEBNext® Multiplex Oligos for Illumina (Methylated Adaptor, Index Primers Set 1) (NEB Cat #E7535S/L) using PCR (16 cycles) with EpiMark® Hot Start Taq DNA Polymerase (NEB Cat #M0490S) following manufacturer instructions (more details can be found on protocols.io).\n\nAfter amplification, prepared libraries were purified using SPRIselect magnetic beads (Cat #B23317). The concentration of prepared libraries was measured using the Thermo Fisher Scientific dsDNA High Sensitivity Assay kit (Cat #Q32851) with an Invitrogen Qubit Fluorometer as well as a NanoDrop ND-1000 spectrophotometer (Cat# E1123552; Thermo Fisher Scientific). Prepared libraries were then sent to Novogene Corporation Inc. (Sacramento, CA, USA) for downstream quality assessment and whole-genome sequencing. Fragment size and concentration were validated with an Agilent Bioanalyzer (Agilent Technologies; Santa Clara, CA, USA). Libraries were sequenced on an Illumina Hi-Seq sequencer to obtain 150 bp paired-end reads.\n\nThe quality of raw reads obtained from the three samples was assessed using FastQC (RRID:SCR_014583) (v.0.11.8) (Andrews, 2010) and MultiQC (RRID:SCR_014982) (v1.9) (Ewels et al., 2016). Adapter sequences and poor-quality bases (Phred <20) were removed using Trim Galore (RRID:SCR_011847) (v0.4.2) (Bolger et al., 2014). The pre-processed reads were then aligned to the soybean reference genome (Gmax_275_v2.0) obtained from Phytozome (Goodstein et al., 2012) using Bismark (RRID:SCR_005604) (v.0.22.3) aligner with default parameters (Krueger and Andrews, 2011). After removing duplicate reads, Bismark (v.0.22.3) (Krueger and Andrews, 2011) was used to detect cytosine methylation at a single-base resolution. Cytosine methylation levels at each region of the sequence were calculated as the number of methylated C vs. total C and T present. To estimate and evaluate false-positive methylation levels and efficiency of cytosine deamination, reads were aligned to the non-methylated controls, soybean chloroplast genome (NC_007942.1), and E. coli non-methylated genomic DNA sequences. Data were visualized with RStudio (RRID:SCR_000432) (v1.1.463) (R Core Team, 2020) implementing the ggplot2 package (RRID:SCR_014601) (v.3.3.5) (Wickham, 2016). The code is available from GitHub and is archived with Zenodo (Wijeratne, 2022).\n\n\nResults and discussion\n\nDespite the utility and growing popularity of WGBS for epigenome analysis in crop plants, relatively little has been done to identify various factors affecting sample preparation and data quality. Cytosine conversion is a crucial step of this process, as partial conversion can lead to false methylation calls. In this survey experiment, we used soybean DNA to prepare samples using three different cytosine conversion protocols. We have evaluated their ease of use, DNA loss during the conversion step, and subsequent data quality. These evaluations shed light on the performance of these three kits and their use for preparing samples to analyze crop plant epigenomes.\n\nEssential factors to consider when selecting a kit are the final yield of libraries and the ease of the protocol. Based on the time required for cytosine conversion steps, the QIAGEN Conversion kit was the fastest method to prepare the whole library (≈ 24 hrs), followed by the NEB EM-seq kit (≈ 26 hrs) and Zymo Conversion kit (≈ 35 hrs). When evaluating the cytosine conversion step, the Zymo Conversion kit needed the longest time of ≈ 16 hrs. However, the QIAGEN Conversion kit and the NEB EM-seq kit required a total of ≈ 5 hrs, which included the post-conversion cleanup step (Tables 1-3).\n\nNEB, New England BioLabs.\n\nFollowing cytosine conversion, the highest recovery of DNA was observed with the QIAGEN conversion kit (100%), followed by the Zymo Conversion (20%) and NEB EM-seq (7.40%) kits (Figure 3). Previously, researchers noted factors that resulted in the reduction of libraries while using SB-based cytosine conversion. In a study conducted by Tanaka and Okamoto (2007), a real-time PCR experiment indicated that DNA degradation during SB conversion occurs primarily due to the hydrolytic reaction or the prolonged exposure to the SB. Additionally, prolonged exposure to SB results in the formation of abasic sites and subsequent DNA strand damage (Tanaka and Okamoto, 2007). An abasic site is a region of DNA that lacks both purine and pyrimidine bases due to DNA damage formed by spontaneous hydrolysis of the N-glycosidic bond (Boiteux and Guillet, 2004). According to the manufacturer's manuals for SB-based conversion kits, DNA is denatured and fragmented before or during cytosine conversion in the thermocycler to chemically convert non-methylated cytosines into uracil. Thus, the prolonged exposure to SB during denaturation, as well as DNA fragmentation, likely contributed to the loss of small DNA fragments during the downstream purification steps, reducing library yield. Conversely, high recovery of DNA when using the QIAGEN conversion kit may be attributed to the presence of DNA protection buffer in the kit (Izzi et al., 2014; Leontiou et al., 2015; Tierling et al., 2018). The drastic loss of DNA with the NEB EM-seq kit may be due to comparatively more purification steps, increasing the chances of DNA loss due to pipetting.\n\nPrepared samples were sequenced to see if the cytosine conversion method had any effect on the sequencing. The initial quality of sequencing data was analyzed based on the following criteria: the total number of sequencing reads obtained, total coverage (X), duplication rate (%), Phred quality score, Per-base sequence, and GC content (%) in the sample. These parameters indicated no major differences among the three samples as discussed below. The number of raw sequencing reads per sample ranged from 33–50 million, with coverage ranging from 20X to 30X (Figure 3). The Phred quality score of each data set was found to be excellent, ranging between 31 to 40 (Figure 4). Based on the per-base sequence plot, thymine was the most abundant base in each library with 50% of all four bases, whereas cytosine was the least abundant (Figure 5). GC content was similar between samples, with the Zymo Research library having the highest level, followed by the QIAGEN and NEB kits (Figure 3). As expected, the per-sequence GC content for each sample was biased due to cytosine conversion into thiamine during PCR amplification. Thus, our data suggest that the cytosine conversion method has little effect on the initial sequence quality.\n\nThe result was obtained using MultiQC. WGBS, whole-genome bisulfite sequencing; EM-seq, enzymatic methylation sequencing.\n\nResults were obtained using MultiQC. NEB, New England BioLabs.\n\nNevertheless, initial data quality may not reflect data utility. Therefore, we mapped reads to the soybean genome to see the mapping efficiency (defined as how many reads can be aligned to the genome proportional to the total reads). Reads generated from all three methods had similar mapping efficiencies. The NEB kit library had the highest mapping efficiency (74.4%), followed by the QIAGEN kit (72.5%) and the Zymo Research kit (70.8%) (Figure 3). The observed mapping efficiency (>70%) is comparable with previously reported efficiencies for cytosine-converted samples (≈ 60–75%) (Hari and Parthasarathy, 2019). These results implied that sequences obtained from all three methods could be aligned correctly to the soybean genome.\n\nWe also calculated the duplication rate for each sample, as a higher rate of duplication distorts the true sequence proportion in a given sample. Duplication rates between 13 and 15% were detected for each library (Figure 3), which is lower than previously reported duplication rates during soybean methylome profiling (Rambani et al., 2015). Duplication can occur for several reasons. For instance, biased PCR enrichment and overamplification of DNA fragments can result in an overrepresentation of library fragments. Duplication can also occur when an identical template binds to numerous clusters on a flow cell. Duplicates of PCR products distort the actual proportion of sequences in the sample (please see more details on the Babraham Institute website).\n\nIt is crucial to analyze the cytosine conversion efficiency during DNA methylation analysis. If a library has low conversion efficiency, it can result in false methylation detection (Singer, 2019). Adding methylated, non-methylated, or both types of genomic controls in a sample can help identify these limitations presented by bisulfite-converted DNA. Here, the cytosine conversion efficiency was calculated using reads aligned to non-methylated E. coli and chloroplast gDNA for each sample. According to these alignments, the bisulfite conversion efficiency of samples ranged between 90 and 99.8% (Figure 3). QIAGEN and Zymo Conversion kits had a similar conversion efficiency of roughly 99% (Figure 3). Similar results were reported for both bisulfite kits in a study conducted on human chromosomal DNA (Tierling et al., 2018). For the NEB EM-seq kit, the cytosine conversion efficiency was found to be 90.9%, around 9% lower than the data obtained from a published study (Figure 3) (Feng et al., 2020). The efficiency and accuracy of a conversion kit are dependent primarily on the PCR cycling procedure, conditions, the length of the desulphonation process, and the addition of reagents to prevent DNA degradation (Izzi et al., 2014; Tierling et al., 2018). In this case, the QIAGEN kit included a DNA protection buffer containing a pH indicator dye to ensure the correct pH for cytosine conversion. Alternatively, the Zymo Research kit protocol distinguishes the alkaline or denaturation step from the conversion step (Izzi et al., 2014). However, it is unclear what contributed to poor cytosine conversion efficiency in the NEB EM-seq kit. It is possible that further optimization of the input DNA quantity is necessary for this kit when it is used with the NEB library preparation kit.\n\nFurthermore, the data were aligned to the soybean reference genome to detect the number of methylated cytosines in each library. The highest number of methylated cytosines in all three contexts (i.e., CpG, CHG, and CHH) was found in the library prepared with the NEB EM-seq kit, with around 1.295 × 109 total methylated cytosines, followed by 1.169 × 109 in the QIAGEN library and 703 million in the Zymo Research library. Moreover, the QIAGEN and Zymo Research libraries displayed a similar distribution of methylated cytosine across contexts. The NEB library showed a distinct distribution compared to the other kits, with an increase in methylated cytosines at CHH context (Figure 6). This can be attributed to the comparatively poor cytosine conversion efficiency, which may have resulted in a high number of erroneously methylated cytosines (Simpson et al., 2017). As expected, the majority of cytosines in each library were methylated in CpG context, followed by CHG and CHH (Figure 6). These data are congruent with a past soybean DNA methylation study, which revealed the greatest levels of methylation in CpG context, followed by CHG and CHH contexts, under normal developmental conditions (Song et al., 2013).\n\nNEB, New England BioLabs.\n\n\nConclusions\n\nThe current work is the first to compare WGBS and EM-seq to analyze DNA methylation in the soybean genome. Here, a survey study was conducted deploying three commercially available DNA methylation detection kits. The results obtained suggested that the QIAGEN kit provided the best DNA yield and number of sequencing reads. The Zymo Research kit demonstrated the best cytosine conversion efficiency and a low duplication rate. Nevertheless, we recommend the NEBNext® Enzymatic Methyl-seq Conversion Module, which was superior to the other kits based upon required DNA input, quality of generated libraries, and hands-on time. Follow-up studies with biological/technical replication are needed to validate the reproducibility and efficiency of the defined conversion kits.",
"appendix": "Data availability\n\nRaw sequencing data are available as follows:\n\nBioProject: Glycine max cultivar: Williams 82 (soybean). Accession number PRJNA902392; https://identifiers.org/bioproject:PRJNA902392 (Arkansas State University, 2022a).\n\nSequence Read Archive: Evaluation of cytosine conversion methods for whole-genome DNA methylation profiling (Run: SRR22331514). Accession number SRX18304416; https://identifiers.org/insdc.sra:SRX18304416 (Arkansas State University, 2022b).\n\nSequence Read Archive: Evaluation of cytosine conversion methods for whole-genome DNA methylation profiling (Run: SRR22331515). Accession number SRX18304415; https://identifiers.org/insdc.sra:SRX18304415 (Arkansas State University, 2022c).\n\nSequence Read Archive: Evaluation of cytosine conversion methods for whole-genome DNA methylation profiling (Run: SRR22331516). Accession number SRX18304414; https://identifiers.org/insdc.sra:SRX18304414 (Arkansas State University, 2022d).\n\nAnalysis code available from: https://github.com/ajwije/DNA_methylation_analysis.git\n\nArchived analysis code at time of publication: https://doi.org/10.5281/zenodo.7328525 (Wijeratne, 2022).\n\nLicense: MIT\n\n\nReferences\n\nAndrews S: FastQC: a quality control tool for high throughput sequence data.2010.\n\nArkansas State University: Glycine max cultivar: Williams 82 (soybean). [Dataset]. BioProject. 2022a.Reference Source\n\nArkansas State University:Evaluation of cytosine conversion methods for whole-genome DNA methylation profiling (Run: SRR22331514). [Dataset]. Sequence Read Archive. 2022b.Reference Source\n\nArkansas State University:Evaluation of cytosine conversion methods for whole-genome DNA methylation profiling (Run: SRR22331515). [Dataset]. 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Bioinformatics. 2011; 27(11): 1571–1572. PubMed Abstract | Publisher Full Text | Free Full Text\n\nLeontiou CA, Hadjidaniel MD, Mina P, et al.: Bisulfite Conversion of DNA: Performance Comparison of Different Kits and Methylation Quantitation of Epigenetic Biomarkers that Have the Potential to Be Used in Non-Invasive Prenatal Testing. PLoS One. 2015; 10(8): e0135058. Publisher Full Text\n\nLi Q, Hermanson PJ, Springer NM:Detection of DNA Methylation by Whole-Genome Bisulfite Sequencing.Lagrimini LM, editor. Maize: Methods and Protocols. Springer;2018; (pp. 185–196). Publisher Full Text\n\nMoore LD, Le T, Fan G: DNA Methylation and Its Basic Function. Neuropsychopharmacology. 2013; 38(1): 23–38. Publisher Full Text\n\nPlongthongkum N, Diep DH, Zhang K: Advances in the profiling of DNA modifications: Cytosine methylation and beyond. Nature Reviews Genetics. 2014; 15(10): 647–661. Publisher Full Text\n\nRambani A, Rice JH, Liu J, et al.: The Methylome of Soybean Roots during the Compatible Interaction with the Soybean Cyst Nematode. Plant Physiology. 2015; 168(4): 1364–1377. PubMed Abstract | Publisher Full Text | Free Full Text\n\nRStudio Team: RStudio: Integrated Development for R. PBC, Boston, MA:RStudio;2020.Reference Source\n\nSchutsky EK, Nabel CS, Davis AKF, et al.: APOBEC3A efficiently deaminates methylated, but not TET-oxidized, cytosine bases in DNA. Nucleic Acids Research. 2017; 45(13): 7655–7665. PubMed Abstract | Publisher Full Text | Free Full Text\n\nSimpson JT, Workman RE, Zuzarte PC, et al.: Detecting DNA cytosine methylation using nanopore sequencing. Nature Methods. 2017; 14(4): 407–410. PubMed Abstract | Publisher Full Text\n\nSinger BD: A Practical Guide to the Measurement and Analysis of DNA Methylation. American Journal of Respiratory Cell and Molecular Biology. 2019; 61(4): 417–428. PubMed Abstract | Publisher Full Text | Free Full Text\n\nSong Q-X, Lu X, Li Q-T, et al.: Genome-Wide Analysis of DNA Methylation in Soybean. Molecular Plant. 2013; 6(6): 1961–1974. PubMed Abstract | Publisher Full Text\n\nTanaka K, Okamoto A: Degradation of DNA by bisulfite treatment. Bioorganic & Medicinal Chemistry Letters. 2007; 17(7): 1912–1915. Publisher Full Text\n\nTierling S, Schmitt B, Walter J: Comprehensive Evaluation of Commercial Bisulfite-Based DNA Methylation Kits and Development of an Alternative Protocol With Improved Conversion Performance. Genetics & Epigenetics. 2018; 10: 1179237X18766097. PubMed Abstract | Publisher Full Text | Free Full Text\n\nVaisvila R, Ponnaluri VKC, Sun Z, et al.: Enzymatic methyl sequencing detects DNA methylation at single-base resolution from picograms of DNA. Genome Research. 2021; 31(7): 1280–1289. PubMed Abstract | Publisher Full Text | Free Full Text\n\nWickham H: ggplot2: Elegant Graphics for Data Analysis. Springer;2016.\n\nWijeratne A: ajwije/DNA_methylation_analysis: v1.0.0 (v1.0.0). Zenodo. [Code].2022. Publisher Full Text\n\nWijesinghe P, Bhagwat AS: Efficient deamination of 5-methylcytosines in DNA by human APOBEC3A, but not by AID or APOBEC3G. Nucleic Acids Research. 2012; 40(18): 9206–9217. PubMed Abstract | Publisher Full Text | Free Full Text"
}
|
[
{
"id": "162649",
"date": "22 Feb 2023",
"name": "Aaron John Stevens",
"expertise": [
"Reviewer Expertise DNA methylation",
"genomics",
"genetics",
"gene structure",
"microbiome",
"rnaseq",
"bioinformatics"
],
"suggestion": "Not Approved",
"report": "Not Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nIn this article the authors evaluate the performance of different DNA methylation profiling techniques that convert cytosine to uracil and assess their validity for high-throughput sequencing. Two commercial sodium bisulfite (SB) conversion kits developed by Qiagen and Zymo were compared with the NEB EM-seq kit (EM) using genomic DNA extracted from soybean leaf. The SB method relies on the deamination of non methylated cytsoine to uracil using the chemical, sodium bisulfite, whereas EM uses the ten-eleven translocation (TET) enzyme to convert 5-mC to 5-hmC, 5-fC, and eventually to 5-caC, protecting methylated cytosines. The APOBEC3A enzyme is then used to deaminate non-methylated cytosines into uracil. EM conversion has been reported to provide superior results compared to SB treatment, as it offers better data, higher coverage, and more reliable detection of methylation states even with extremely low levels of DNA input. Presumably the main point of difference is that this article investigates plant epigenomes using soybean DNA.\nThe QIAGEN Conversion kit outperformed the other kits in terms of time requirement and DNA recovery. The NEB EM-seq kit resulted in the lowest DNA recovery, possibly due to more purification steps increasing the chances of DNA loss. However, all three kits yielded quality sequencing data with no major differences in total number of sequencing reads, total coverage, duplication rate, Phred quality score, per-base sequence, and GC content. The results suggest that the cytosine conversion method has little effect on the initial sequence quality, and factors such as time requirement and DNA recovery should be considered when selecting a kit for preparing samples for WGBS analysis of crop plant epigenomes.\nMajor concerns:\nThis is a superficial analysis and it does not appear that the authors have considered the use of replicates and instead present the results from a single run of each method. Without replicates there is no way to measure intra kit variation rates, which effectively renders the results meaningless. I have performed hundreds of bisulfite conversions with the zymo kit and will frequently see variation in the conversion ratio, the quality and the yield of DNA between samples from the same input DNA quality and content.\n\nHave the authors measured returned yield of DNA through the kit against the input amount? This is very important consideration especially when sample is limited.\n\nThe figures and figure caption are not presented in a manner appropriate for scientific publication. For example Figure 3 should be labelled as figure 3A, B, C with a full caption and no legend.\n\nAuthors state several experiments in their methods that they do not include in their results. E.g. gel electrophoresis and that the DNA quantity and quality were measured (neither appear to be stated). However “library recovery” is stated with an inadequate description of what this relates to. Is this the DNA amount measured pre and post conversion? Where is the raw data relating to this?\n\nWhere is the fragment size information prior to sequencing. Should DNA fragment size have been compared prior to the bisulfite conversion also?\nMinor concerns:\nThe first subheading in results/Discussion is a full summary of the section.\n\nTable 3 should be in methods not in 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? 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? No\n\nAre the conclusions drawn adequately supported by the results? No",
"responses": []
},
{
"id": "167362",
"date": "04 Apr 2023",
"name": "Sadaruddin Chachar",
"expertise": [
"Reviewer Expertise Epigenetics",
"Genomics",
"Rice",
"Photosynthesis",
"DNA methylation",
"Histone posttranslational modifications",
"RNA-seq"
],
"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\nOverall, this research article represents an interesting investigation on “Evaluation of cytosine conversion methods for whole-genome DNA methylation profiling”, to assess the performance of various DNA methylation profiling techniques and evaluate their validity for high-throughput sequencing. Abstract seems logical providing the concise summary of the findings, the introduction provides sufficient background of the study, while the methods are not generally appropriate for the experiments conducted. The analysis and results presented in figures seem logical while interpretation is supported by results. Moreover, the results are clearly described, making the manuscript easily understandable for readers. All the comments and remarks are given below.\nIn the first line of abstract, the correct definition of DNA methylation is the addition of a methyl group to the cytosine base in the DNA molecule, not the removal or addition of methyl groups to cytosine bases.\nDiscuss the limitations of the previous works as a motivation of the current study.\nWhy were these kits selected? As there are numerous other kits available as well, the justification for selecting these kits is missing.\nIn the last paragraph of introduction, authors have mentioned that “However, optimized sample preparation protocols are only available for a handful of species and are missing for many important crop plants”. While they have used only soyabean in this study. So how can they justify that the method optimized in this study for soyabean can work best for other species as well?\nAuthors have not mentioned how many samples per method they used and how many replicates of each method were used.\nIn material and methods, authors have measured purity of DNA by Nanodrop and quality and size of DNA using gel electrophoresis, while no such data is given in manuscript. There are numerous other methods mentioned while no results are given for them.\nFigure 5 is of low quality and hard to read, its quality needs to be improved.\n\nIs the work 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? No\n\nAre the conclusions drawn adequately supported by the results? Yes",
"responses": []
}
] | 1
|
https://f1000research.com/articles/11-1450
|
https://f1000research.com/articles/11-1449/v1
|
07 Dec 22
|
{
"type": "Research Article",
"title": "Risk factors and mortality outcomes of extended-spectrum beta-lactamase producing Escherichia coli bacteremia: A retrospective cohort study from two Indonesian referral hospitals",
"authors": [
"Masra Lena Siregar",
"Erni Juwita Nelwan",
"Eppy .",
"Budi Haryanto",
"Nelly Puspandari",
"Robert Sinto",
"Leonard Nainggolan",
"Maruhum Bonar",
"Hamzah Shatri",
"Erni Juwita Nelwan",
"Eppy .",
"Budi Haryanto",
"Nelly Puspandari",
"Robert Sinto",
"Leonard Nainggolan",
"Maruhum Bonar",
"Hamzah Shatri"
],
"abstract": "Background: Bacteremia caused by extended-spectrum beta-lactamase (ESBL)-producing Escherichia coli (E. coli) can lead to bloodstream infection and subsequent sepsis which increases morbidity and mortality. Evaluation of risk factors of infection by ESBL-producing E. coli is important as it can decrease inappropriate antibiotic use and mortality rates. This study aimed to identify the risk factors and mortality of bacteremia caused by ESBL-producing E. coli. Methods: This retrospective cohort study included inpatients with confirmed E. coli blood culture examinations from two referral hospitals in Jakarta, Indonesia. Data suspected as risk factors for ESBL-producing E. coli bacteremia (utilization of medical devices, age, Charlson Comorbidity Index, history of hospitalization, and history of antibiotic therapy) were collected for analysis. Clinical profiles and independent risk factors of ESBL-producing E. coli bacteremia associated mortality were also evaluated. Results: A total of 116 subjects were analyzed with 81% aged ≥18 years old. The most common source of infection was the gastrointestinal and intra-abdominal tracts. Malignancy as comorbidity was present in 46.6% subjects. Significant risk factors for developing ESBL-producing E. coli bacteremia were history of antibiotic therapy and utilization of medical devices. The proportion of mortality in ESBL-producing E. coli bacteremia was 55.7% with age and sepsis as its independent risk factors. Conclusions: History of antibiotic therapy and utilization of medical devices were significant risk factors for ESBL-producing E. coli bacteremia. The proportion of mortality in ESBL-producing E. coli bacteremia patients was 55.7% with its independent risk factors being age and sepsis.",
"keywords": [
"bacteremia",
"Escherichia coli",
"extended-spectrum beta-lactamase",
"risk factor"
],
"content": "Introduction\n\nBacteremia or the presence of bacteria in blood can develop into bloodstream infection which, if left untreated, can lead to death from sepsis or septic shock.1–3 It is very difficult to find bacteria in blood unless in severe cases and it may not correlate with clinical features at the time of infection.2,4 In several studies, bacteremia was most often caused by gram-negative bacteria with Escherichia coli (E. coli) being the most common etiology with an incidence rate of 50 to 60 cases per 100,000 population.5–7\n\nBased on the World Health Organization (WHO) global priority pathogens list of antibiotic-resistant bacteria (https://www.who.int/news/), extended-spectrum beta-lactamase (ESBL)-producing Enterobacteriaceae is included in the first priority (critical) group and ESBL-producing E. coli is the most reported.8 ESBL is an enzyme produced by certain bacteria capable of hydrolyzing broad-spectrum beta-lactam antibiotics such as penicillin, first-, second-, and third-generation cephalosporins (ceftriaxone, cefotaxime, and ceftazidime), and monobactams (aztreonam), causing multidrug resistance (MDR).8,9\n\nIn Indonesia, the prevalence of ESBL from various isolates is fairly high at 60–85%.10,11 The “One Health” surveillance research in public health, agriculture, and animal and livestock sectors conducted by the Indonesian Ministry of Health Research and Development found that the proportion of ESBL-producing E. coli bacteremia reached 57.7%.12 Meanwhile, the mortality rate of E. coli bacteremia in several studies ranged from 18.2–30.6%.13–15 Most bacteremic patients develop sepsis and the excessive use of antibiotics can cause antibiotic resistance, therefore prolonging the length of hospital stay which may in turn lead to increased mortality.13,14,16\n\nSeveral studies have identified several risk factors for ESBL-producing E. coli and associated mortality.16–19 Demographic factors, antibiotic resistance patterns, sources of infection, and disease severity in each hospital are deemed necessary to be evaluated as risk factors for ESBL-producing E. coli and associated mortality that clinicians concern themselves with these factors in the management of patients with infections caused by antibiotic-resistant bacteria.\n\n\nMethods\n\nAn observational retrospective cohort study was conducted using the medical records of all inpatients from January to December 2019 at two referral hospitals in Jakarta, Indonesia, namely Cipto Mangunkusumo National Hospital and Persahabatan Central General Hospital. This study was approved by the Ethics Commission of The National Institute of Health Research and Development (LB.02.01/2/KE.184/2018) on 8 May 2018, the Health Research Ethics Committee of the Faculty of Medicine, Universitas Indonesia-Cipto Mangunkusumo National Hospital (KET-169/UN2.F1/ETIK/PPM.00.02/2022) on 21 February 2022, and the Health Research Ethics Committee of Persahabatan Central General Hospital (42/KEPK-RSUPP/07/2018) on 31 July 2018.\n\nThe target population was patients with positive E. coli blood cultures which were taken once during hospitalization in all age groups. Patients who were treated for less than 48 hours, had negative E. coli blood cultures or with incomplete medical record data were excluded. Positive E. coli blood cultures were phenotypically differentiated into ESBL- and non-ESBL-producing colonies using the VITEK®2 compact system algorithm (bioMérieux, France). Patient characteristics, length of stay, source of infection, comorbidity, and several risk factor variables for ESBL-producing E. coli infection including age, history of antibiotic therapy, utilization of medical devices, Charlson Comorbidity Index (CCI), and history of hospitalization were extracted from medical records. Further analysis was conducted for patients with ESBL-producing E. coli bacteremia. An effort was made to avoid missing data bias by excluding patients with incomplete records and differential measurement bias was addressed by uniformizing confirmation tests between all patients.\n\nElaboration on the process of subject inclusion is available in the Underlying data (Supplementary Figure 1).20 The sample size was determined by calculating the formula for comparing the difference between two independent proportions. A total of 118 subjects were determined to be needed for adequate hypothesis testing. The details are available in the Underlying data (Supplementary Figure 2).20\n\nThe basic and clinical characteristics of the research subjects are presented in Table 1 and as underlying data.20 Categorical data are presented as proportions. The data are presented as means and standard deviations if they were normally distributed and median and minimum-and-maximum values if not. Risk factors for acquiring ESBL-producing E. coli bacteremia and risk factors for mortality in patients with such conditions were analyzed. Bivariate analysis using Chi-square or Fisher’s exact test was performed to examine the significance of the odds ratio (OR). A multivariate logistic regression test was subsequently performed to identify significantly contributing factors for variables with a bivariate analysis p value of less than 0.25. Statistical analyses were conducted with IBM SPSS Statistics version 20 (RRID:SCR_002865).\n\n\nResults\n\nA total of 116 subjects were analyzed in this study. The basic characteristics of patients with E. coli bacteremia are shown in Table 1. The most common source of infection was the gastrointestinal and intra-abdominal tract. More than half of the patients had a history of hospitalization and a history of antibiotic therapy, mostly with cephalosporins. Most patients were treated with empirical antibiotic therapy with the same drug group. Malignancy was the most frequent comorbidity. Most were found to have CCI scores of greater than two. Medical devices were commonly utilized among patients with urinary catheters and nasogastric tubes being the most common. Most had sepsis and nearly half was lethal.\n\nBivariate analysis showed significant statistical differences (p <0.05) in crude OR for ESBL-producing E. coli bacteremia between age, utilization of medical devices, CCI scores, and history of antibiotic therapy. Variables with p-values of <0.25 were included for multivariate logistic regression. The analysis yielded statistically significant risk from utilization of medical devices and history of antibiotic therapy. Specifically, it showed that history of antibiotic therapy and the use of medical devices increases the risk of developing ESBL-producing E. coli bacteremia by 2.78 and 3.21 times respectively (Table 2).\n\nThe proportion of mortality between patients with ESBL- and non-ESBL-producing E. coli bacteremia was 55.7% and 38.2% respectively (p = 0.059). Patients infected by the ESBL-producing strain were further analyzed for mortality risk factors (Table 3). Out of all the variables, only sepsis produced a statistically significant difference in mortality (p = 0.011) in the bivariate analysis. Furthermore, multivariate logistic regression analysis of the eligible variables (age, length of stay, utilization of mechanical ventilator, empirical antibiotic therapy, and sepsis) showed that only age (OR 15.0; 95% CI 1.54 to 146.02; p = 0.020) and sepsis (OR 6.5; 95% CI 1.91 to 22.16; p = 0.003) produced statistically significant differences in mortality.\n\n\nDiscussion\n\nIn this study, the proportion of patients with ESBL-producing E. coli bacteremia was 52.58%. This result was higher compared to some other studies.15,16,18 Both ESBL and non-ESBL-producing E. coli bacteremia can occur in various age groups.21,22 Epidemiological studies reported the proportion of E. coli bacteremia ranging from 18–55% in all age groups.23 A 2017 study by Nivesvivat et al. in a Thai tertiary care hospital found that the prevalence of ESBL-producing Enterobacteriaceae bacteremia in children has tended to increase since 2010.24 Bacteremia by ESBL-producing Enterobacteriaceae significantly associated with higher mortality than its non-ESBL-producing counterpart. The study found that this condition was significantly more prevalent in children with comorbidities such as chronic illnesses.24 The median age of subjects in our study was 48 years (range 0–89 years) with males being the predominant gender (52.6%). Around 18.9% of patients were less than 18 years old, which ranged from neonates to adolescents. Our findings were similar to previous studies that described the age distribution of bacteremic patients which ranged from pediatric to elderly patients.7,22,25\n\nAccording to the multivariate analysis, patients aged 18 years or older were more than twice as likely to experience ESBL-producing E. coli bacteremia though this was not statistically significant. Neonates and the elderly are groups susceptible to infection. Children tend to experience more severe forms of bacteremia due to their immature immune systems. Neonates can be exposed to bacteria by vertical transmission during pregnancy or from the mucosa of the birth canal, especially in the presence of bacterial colonization. Furthermore, E. coli strains found in children aged less than three months were reported to be genotypically more virulent than those found in adults.26 Meanwhile, diminished immune responses along with the presence of other risk factors in geriatric patients increase the risk of MDR infection.\n\nThe most common sources of infection were the gastrointestinal and intra-abdominal tracts (37.1%). No significant difference was observed in the proportion of infection sources between the ESBL and non-ESBL groups. Many other studies also found gastrointestinal tract as a source of infection.19,27,28 E. coli is a gram-negative bacteria found in the normal intestinal flora of healthy individuals which may become pathogens if present in an excessive amount.29,30 Transmission of E. coli can occur by direct contact or fecal-oral through contaminated food and/or water. Hence, some types of E. coli can cause diarrhea, gastrointestinal infections, urinary tract infections, respiratory tract infections, and other diseases.22,29 Therefore, the source of the infection site for E. coli bacteremia can come from various organ systems. The most common sources of infection related to mortality from ESBL-producing E. coli bacteremia is from the gastrointestinal and respiratory tracts. Different results were reported in several other studies, the urinary tract was the most common source of infection associated with mortality in E. coli ESBL bacteremia.13,18,31\n\nIn this study, history of antibiotic therapy was found in as many as 63.8% of patients with E. coli bacteremia and 77.1% ESBL-producing E. coli bacteremia. Previous studies reported a history of antibiotic therapy in 40 to 80% of ESBL-producing E. coli bacteremia.17–19 Additionally, the multivariate analysis showed that a history of antibiotic therapy was a significantly associated risk factor for ESBL-producing E. coli bacteremia. Other studies have also demonstrated the same results.16–18,28 History of antibiotic therapy was reported to be a significant risk factor for ESBL-producing bacteremia in adults but not in children.18,19,21,32 Furthermore, the most widely used type of antibiotic is the beta-lactam group, especially cephalosporins, which are widely used as an empirical antibiotic due to their broad spectrum of activity against gram-positive and negative bacteria and even some resistant strains.33 Inadequate empirical antibiotic therapy during treatment in the intensive care unit (ICU) or in the regular ward may lead to colonization of ESBL-producing bacteria and potentially lead to antibiotic resistance.34\n\nMany studies have reported the use of medical devices as a risk factor for MDR bacteremia.16,18,31,35 In this study, utilization of medical devices was also significant as a risk factor for ESBL-producing E. coli bacteremia. Medical devices are thought to be a mediator for the entry of bacteria into the blood which can then progress to bacteremia. Trauma to the skin and mucosal surfaces causes epithelial antigen-presenting cells (such as dendritic cells or Langerhans cells) to capture bacterial antigens, they then migrate to lymph nodes, and are presented to T-lymphocytes.1,2\n\nThe Charlson Comorbidity Index is commonly used for the assessment of risk factors and predictors of prognosis.36 Although not a risk factor, we found a statistically significant difference was observed in the OR between ESBL and non-ESBL-producing E. coli bacteremia patients with CCI scores of greater than two. Nguyen et al. and Kaya et al. also reported that comorbidity was not proven as a risk factor for ESBL-producing E. coli bacteremia.16,27 In this study, malignancy was the most common comorbidity. Patients with malignancies are susceptible to infection by ESBL-producing E. coli, a condition which may increase mortality in such a population.37 Patients are generally immunosuppressed due to the underlying disease and the treatment of the disease itself.38 The use of medical devices and invasive procedures further puts patients with malignancy in higher risk of MDR infection.3,22,23 The innate and adaptive immune systems play a major role in microbial clearance along with the spleen and liver which filter bacteria entering blood circulation. The introduction of bacteria to the bloodstream may resolve spontaneously or develop into a bloodstream infection that leads to sepsis.1–3 Coexistence of bacteremia with malignancy may result in a vicious cycle as it renders patients with malignancy in a constant immunosuppressive and hyperinflammatory state, which consequently affects the innate and acquired immune systems even in the recovery period.38\n\nWe also found that history of hospitalization was not a statistically significant risk factor for ESBL-producing E. coli bacteremia. However, different results were reported by Xiao et al. who found that a history of hospitalization in the past three months doubled the risk of developing ESBL-producing E. coli bacteremia.28 A study conducted by Su et al. reported that elderly patients with a history of hospitalization were more than three times as likely to be infected by ESBL-producing E. coli or K. pneumoniae bacteremia even though they had been discharged more than 360 days ago.39 The use of urinary catheters is cited as one of the most common sources of infection and medium for bacterial colonization. Utilization of such medical devices is often associated with a history of hospitalization.29,30,37 Moreover, patients with a history of hospitalization are at a higher risk of bacterial colonization and subsequent MDR bacteremia due to exposure from MDR bacteria at the ICU or regular ward and previous administration of antibiotics. The aforementioned factors along with the fact that our study was conducted at two referral hospitals predominated by patients with malignancies who have had a history of multiple hospitalizations (and consequently have already been exposed to MDR bacteria several times) may provide a plausible explanation for our findings.\n\nThere was no significant difference in the proportion of mortality between subjects with ESBL-producing and non-ESBL-producing bacteremia. Sianipar et al. previously reported no significant difference in the proportion of mortality between ESBL- (30.6%) and non-ESBL-producing (22.2%) E. coli bacteremia.14 Kang et al. reported a mortality proportion of 25.6% in ESBL-producing E. coli bacteremia patients with no meaningful difference between the groups for gender and age.15 We also found that administration of empirical antibiotic therapy did not have a significant difference in the incidence of mortality. Research by To et al. demonstrated that administration of carbapenem and non-carbapenem empirical antibiotic therapy had no significant effect on 30-day mortality.31 Appropriate and compatible definitive antibiotic therapy is required in bloodstream infections if proven to be caused by MDR bacteria. Delay in antibiotic administration is associated with increased morbidity and mortality.40 Therefore, prompt identification of the offending bacteria in bloodstream infections is crucial.\n\nThe multivariate analysis indicated that age and the presence of sepsis are significant risks for mortality in patients with ESBL-producing E. coli bacteremia. Rodriguez et al. also reported that patients with sepsis due to ESBL-producing E. coli are over three times and over four times more likely to experience 30-day and 14-day mortality respectively.18 Other factors such as length of stay, use of mechanical ventilators, and empirical antibiotic therapy were not found to be statistically significant, but these factors are often associated with other factors including history of antibiotic therapy and history of hospitalization. The effect of the presence of ESBL-producing strains of E. coli on bacteremia mortality outcomes is still controversial. Several studies have found an association between infection by ESBL-producing E. coli and mortality while another study reported contradictory results showing no difference in mortality between the two patient groups.7\n\nAcross the numerous studies that have been conducted to assess the risk factors for ESBL-producing E. coli bacteremia, there exist differences in demography, bacterial patterns, and causes of bacteremia in each hospital. This study, which is part of the “One Health” study conducted by the Centre for Biomedical Research and Development and Basic Health Technology of the Indonesian Ministry of Health, provides a well-detailed investigation of risk factors for ESBL-producing E. coli bacteremia in two referral hospitals in Indonesia.12\n\nSeveral limitations are present in this study. First, the retrospective nature of this study renders it to rely on secondary data obtained from the paper medical records of the two hospitals previously mentioned. Therefore, the occurrence of missing data was unavoidable and perfect representation of the data would be difficult to achieve. Second, several factors such as an onset of bacteremia before positive culture results, risk factors for mortality such as SOFA score, duration of empirical antibiotic therapy, and the type and duration of definitive antibiotic therapy were not accounted for in this study. Third, the ESBL-producing strains were only identified phenotypically and not genotypically. Nevertheless, the results of this study can be applied clinically and are also useful in stratifying risk factors for bacteremia caused by ESBL-producing E. coli. Clinicians should stay vigilant for these risk factors to limit mortality and morbidity from ESBL-producing E. coli.\n\n\nConclusions\n\nTo summarize, history of antibiotic therapy and utilization of medical devices were significant risk factors for ESBL-producing E. coli bacteremia. The proportion of mortality in ESBL-producing E. coli bacteremia patients was 55.7% with age and sepsis being its independent risk factors. Some of the other risk factors were found to not be significantly associated with the incidence of ESBL-producing E. coli infection. Nevertheless, it is still necessary for clinicians to identify these risk factors to stratify patients at risk of infection by MDR bacteria. Hence, the identification of risk factors for MDR bacterial infection remains an important issue as their current treatment options are mostly limited to carbapenems.40\n\n\nConsent\n\nWritten informed consent for publication of the patients’ details was obtained from the patients and guardians of the patients.",
"appendix": "Data availability\n\nMendeley Data: Underlying data for ‘Risk factors and mortality outcomes of extended-spectrum beta-lactamase producing-Escherichia coli bacteremia: A retrospective cohort study from two Indonesian referral hospitals’. https://www.doi.org/10.17632/rnk7yhcvmg.2 20\n\nThis project contains the following underlying data:\n\n- Data file 1: Dataset of all patients whose blood culture was positive for E. coli.xlsx\n\n- Data file 2: Dataset of all patients confirmed to be infected by ESBL-producing E. coli.xlsx\n\n- Data file 3: Dataset of all patients whose blood culture was positive for E. coli.sav\n\n- Data file 4: Dataset of all patients confirmed to be infected by ESBL-producing E. coli.sav\n\n- Supplementary Figure 1: Flow diagram of subject inclusion.docx\n\n- Supplementary Figure 2: Determination of sample size and results.docx\n\nMendeley Data: STROBE checklist for ‘Risk factors and mortality outcomes of extended-spectrum beta-lactamase producing-Escherichia coli bacteremia: A retrospective cohort study from two Indonesian referral hospitals’. https://www.doi.org/10.17632/rnk7yhcvmg.2 20\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0)\n\n\nAcknowledgments\n\nWe are grateful to Cipto Mangunkusumo Hospital and Persahabatan Central General Hospital for their cooperation in providing the data for research and especially to the Centre for Research and Development of Biomedical and Basic Health Technology, Ministry of Health, Jakarta, Indonesia, which has allowed us to continue this research.\n\n\nReferences\n\nTimsit JF, Ruppé E, Barbier F, et al.: Bloodstream infections in critically ill patients: an expert statement. Intensive Care Med. 2020; 46(2): 266–284. PubMed Abstract | Publisher Full Text | Free Full Text\n\nChristaki E, Giamarellos-Bourboulis EJ: The complex pathogenesis of bacteremia: From antimicrobial clearance mechanisms to the genetic background of the host. Virulence. 2014; 5(1): 57–65. PubMed Abstract | Publisher Full Text | Free Full Text\n\nBharadwaj R, Bal A, Kapila K, et al.: Blood stream infections. Biomed. Res. Int. 2014; 2014: 10–12. PubMed Abstract | Publisher Full Text | Free Full Text\n\nKomori A, Abe T, Kushimoto S, et al.: Characteristics and outcomes of bacteremia among ICU-admitted patients with severe sepsis. Sci. Rep. 2020; 10(1): 2983–2988. PubMed Abstract | Publisher Full Text | Free Full Text\n\nLaupland KB: Incidence of bloodstream infection: A review of population-based studies. Clin. Microbiol. Infect. 2013; 19(6): 492–500. Publisher Full Text\n\nKern WV, Rieg S: Burden of bacterial bloodstream infection d a brief update on epidemiology and significance of multidrug-resistant pathogens. Clin. Microbiol. Infect. 2020; 26(2): 151–157. Publisher Full Text\n\nAbernethy J, Guy R, Sheridan EA, et al.: Epidemiology of Escherichia coli bacteraemia in England: results of an enhanced sentinel surveillance programme. J. Hosp. Infect. 2017; 95: 365–375. PubMed Abstract | Publisher Full Text\n\nGhafourian S, Sadeghifard N, Soheili S, et al.: Extended spectrum beta-lactamases: Definition, classification and epidemiology. Curr. Issues Mol. Biol. 2014; 17(1): 11–22. Publisher Full Text\n\nCastanheira M, Simner PJ, Bradford PA: Extended-spectrum β-lactamases: an update on their characteristics, epidemiology and detection. JAC-Antimicrobial Resist. 2021; 3(3): dlab092. PubMed Abstract | Publisher Full Text | Free Full Text\n\nAnggraini D, Sholihin UH, Savira M, et al.: Prevalensi dan Pola Sensitivitas Enterobacteriaceae Penghasil ESBL di RSUD Arifin Achmad Pekanbaru. J Kedokt Brawijaya. 2018 Feb 28; 30(1): 47–52. Publisher Full Text Reference Source\n\nHayati Z, Rizal S, Putri R: Isolation Of Extended-Spectrum B-Lactamase (ESBL) Producing Escherichia coli and Klebsiella pneumiae From Dr. Zainoel Abidin General Hospital, Aceh. Int J Trop Vet. Biomed. Res. 2019 May 1; 4(1): 16–22. Publisher Full Text Reference Source\n\nPuspandari N, Sunarno S, Febrianti T, et al.: Extended spectrum beta-lactamase-producing Escherichia coli surveillance in the human, food chain, and environment sectors: Tricycle project (pilot) in Indonesia. One Heal. 2021; 13: 100331. PubMed Abstract | Publisher Full Text | Free Full Text\n\nAbernethy JK, Johnson AP, Guy R, et al.: Thirty day all-cause mortality in patients with Escherichia coli bacteraemia in England. Clin. Microbiol. Infect. 2015; 21(3): 251, e1–e8. Publisher Full Text\n\nSianipar O, Asmara W, Dwiprahasto I, et al.: Mortality risk of bloodstream infection caused by either Escherichia coli or Klebsiella pneumoniae producing extended-spectrum β-lactamase: A prospective cohort study. BMC. Res. Notes. 2019; 12(1): 1–7. PubMed Abstract | Publisher Full Text | Free Full Text\n\nKang CI, Kim SH, Wan BP, et al.: Bloodstream infections due to extended-spectrum β-lactamase-producing Escherichia coli and Klebsiella pneumoniae: Risk factors for mortality and treatment outcome, with special emphasis on antimicrobial therapy. Antimicrob. Agents Chemother. 2004; 48(12): 4574–4581. PubMed Abstract | Publisher Full Text | Free Full Text\n\nNguyen M-L, Toye B, Kanji S, et al.: Risk Factors for and Outcomes of Bacteremia Caused by Extended-Spectrum ß-Lactamase– Producing Escherichia coli and Klebsiella Species at a Canadian Tertiary Care Hospital. Can. J. Hosp. Pharm. 2015 Apr 28; 68(2): 136–143. PubMed Abstract | Publisher Full Text | Free Full Text Reference Source\n\nWu U, Yang C, Chen W, et al.: Risk Factors for Bloodstream Infections due to Extended-spectrum beta-lactamase-producing Escherichia coli. J. Microbiol. Immunol. Infect. 2010; 43(4): 310–316. Publisher Full Text\n\nRodríguez-Baño J, Picón E, Gijón P, et al.: Risk factors and prognosis of nosocomial bloodstream infections caused by extended-spectrum-β-lactamase-producing Escherichia coli. J. Clin. Microbiol. 2010; 48(5): 1726–1731. PubMed Abstract | Publisher Full Text | Free Full Text\n\nDenis B, Lafaurie M, Donay JL, et al.: Prevalence, risk factors, and impact on clinical outcome of extended-spectrum beta-lactamase-producing Escherichia coli bacteraemia: A five-year study. Int. J. Infect. Dis. 2015; 39: 1–6. PubMed Abstract | Publisher Full Text\n\nSiregar ML, Nelwan E, Eppy E, et al.: Underlying data for “Risk Factors and Mortality Outcomes of Extended-Spectrum Beta-Lactamase-Producing Escherichia coli Bacteremia: A Retrospective Cohort Study from Two Indonesian Referral Hospitals.”. Mendeley Data. 2022; v2. Publisher Full Text\n\nTsai W, Hung C, Chen H, et al.: Extended-spectrum β-lactamase-producing Escherichia coli bacteremia: Comparison of pediatric and adult populations. J. Microbiol. Immunol. Infect. 2018 Dec; 51(6): 723–731. Publisher Full Text Reference Source\n\nBou-Antoun S, Davies J, Guy R, et al.: Descriptive epidemiology of Escherichia coli bacteraemia in England, April 2012 to March 2014. Eurosurveillance. 2016 Sep 1; 21(35): 303209. Publisher Full Text\n\nBonten M, Johnson JR, Van Den Biggelaar AHJ , et al.: Epidemiology of Escherichia coli Bacteremia: A Systematic Literature Review. Clin. Infect. Dis. 2021; 72(7): 1211–1219. PubMed Abstract | Publisher Full Text\n\nNivesvivat T, Piyaraj P, Thunyaharn S, et al.: Clinical epidemiology, risk factors and treatment outcomes of extended-spectrum beta-lactamase producing Enterobacteriaceae bacteremia among children in a Tertiary Care. BMC. Res. Notes. 2018; 11(624): 1–5. PubMed Abstract | Publisher Full Text | Free Full Text\n\nDaga AP, Koga VL, Soncini JGM, et al.: Escherichia coli Bloodstream Infections in Patients at a University Hospital: Virulence Factors and Clinical Characteristics. Front. Cell. Infect. Microbiol. 2019 Jun 6; 9(191): 1–10. Publisher Full Text\n\nBurdet C, Denamur E, Stéphane B, et al.: Escherichia coli Bacteremia in Children. Pediatr. Infect. Dis. J. 2014; 33(8): 872–879. Publisher Full Text\n\nKaya O, Akcam FZ, Gonen I, et al.: Risk factors for bacteremia due to extended-spectrum beta-lactamase-producing Escherichia coli in a Turkish hospital. J. Infect. Dev. Ctries. 2013; 7(7): 507–512. Publisher Full Text\n\nXiao Y, Hang Y, Chen Y, et al.: A Retrospective Analysis of Risk Factors and Patient Outcomes of Bloodstream Infection with Extended-Spectrum β-Lactamase-Producing Escherichia coli in a Chinese Tertiary Hospital. Infect Drug Resist. 2020 Nov; 13: 4289–4296. PubMed Abstract | Publisher Full Text | Free Full Text Reference Source\n\nKaranika S, Karantanos T, Arvanitis M, et al.: Fecal Colonization With Extended-spectrum Beta-Lactamase–Producing Enterobacteriaceae and Risk Factors Among Healthy Individuals: A Systematic Review and Metaanalysis. Clin. Infect. Dis. 2016; 63(3): 310–318. PubMed Abstract | Publisher Full Text\n\nKawamura K, Nagano N, Suzuki M, et al.: ESB-producing Escherichia coli and Its Rapid Rise among Healthy People. Food Saf. 2017; 5(4): 122–150. PubMed Abstract | Publisher Full Text | Free Full Text\n\nTo KKW, Lo WU, Chan JFW, et al.: Clinical outcome of extended-spectrum beta-lactamase-producing Escherichia coli bacteremia in an area with high endemicity. Int. J. Infect. Dis. 2013; 17(2): e120–e124. PubMed Abstract | Publisher Full Text\n\nAlkan F, Salih G, Bayram N, et al.: Risk factors for bacteremia with extended-spectrum β-lactamase production in positive Escherichia coli bacteremia in a pediatric setting. Am. J. Infect. Control. 2017; 45: 1414–1415. PubMed Abstract | Publisher Full Text\n\nKim JY, Yum Y, Joo HJ, et al.: Impact of antibiotic usage on extended-spectrum β-lactamase producing Escherichia coli prevalence. Sci. Rep. 2021; 11(13024): 13024–13027. PubMed Abstract | Publisher Full Text | Free Full Text\n\nHarris AD, McGregor JC, Johnson JA, et al.: Risk factors for colonization with extended-spectrum β-lactamase- producing bacteria and intensive care unit admission. Emerg. Infect. Dis. 2007; 13(8): 1144–1149. PubMed Abstract | Publisher Full Text | Free Full Text\n\nRuiz-Giardin JM, Ochoa Chamorro I, Velázquez Riós L, et al.: Blood stream infections associated with central and peripheral venous catheters. BMC Infect. Dis. 2019; 19(1): 841–849. PubMed Abstract | Publisher Full Text | Free Full Text\n\nCharlson ME, Pompei P, Ales KL, et al.: A new method of classifying prognostic comorbidity in longitudinal studies: Development and validation. J. Chronic Dis. 1987 Jan; 40(5): 373–383. PubMed Abstract | Publisher Full Text Reference Source\n\nAlevizakos M, Karanika S, Detsis M, et al.: Colonisation with extended-spectrum β-lactamase-producing Enterobacteriaceae and risk for infection among patients with solid or haematological malignancy: a systematic review and meta-analysis. Int. J. Antimicrob. Agents. 2016 Dec; 48(6): 647–654. Publisher Full Text Reference Source\n\nGudiol C, Albasanz-puig A, Cuervo G, et al.: Understanding and Managing Sepsis in Patients With Cancer in the Era of Antimicrobial Resistance. Front. Med. 2021; 8(636547): 1–15. PubMed Abstract | Publisher Full Text | Free Full Text\n\nSu Y, Kung L, Lee C, et al.: Antimicrobial-Resistant Bacteremia in the Elderly: Risk of Previous. Int. J. Gerontol. 2017; 11(1): 27–30. Publisher Full Text\n\nPana ZD, Zaoutis T: Treatment of extended-spectrum β-lactamase-producing enterobacteriaceae (ESBLs) infections: What have we learned until now?. F1000Res. 2018; 7: 1–9. PubMed Abstract | Publisher Full Text | Free Full Text"
}
|
[
{
"id": "176826",
"date": "18 Jul 2023",
"name": "Matteo Boattini",
"expertise": [
"Reviewer Expertise Antimicrobial resistance",
"diagnostic stewardship",
"sepsis"
],
"suggestion": "Not Approved",
"report": "Not Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nI read with interest the paper by Nelwan et al. They investigated risk factors for ESBL-producing E. coli bloodstream infection and mortality in a small cohort of patients with bacteremia caused by the same pathogen. The topic is of interest for a general readership even though it is not new and there are already validated scores in the literature with good discriminative ability (please see Palacios-Baena et al doi: 10.1093/jac/dkw5131).\nNotwithstanding the small sample and the retrospective design, the study has several flaws:\nThe variables considered have not been defined (which medical devices are considered? What is meant by a mechanical ventilator? The history of hospitalisation and antibiotherapy consider what time interval? What is the definition of sepsis? etc etc.)\n\nDoes the study identify risk factors for bloodstream infection and mortality for patients who had received appropriate empirical therapy, or not?\n\nWhy did the authors not use an index of the severity of bacteremia such as the Pitt score?\n\nFrom a prognostic point of view, it does not make much sense to use the length of stay in the analyses as it can only be known at the end of the event.\n\nI think that the authors have embarked on a study dealing with an already much debated topic and that the power of their study, even considering its many limitations, is not such as to add much of significance.\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Partly\n\nAre sufficient details of methods and analysis provided to allow replication by others? No\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nPartly\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Partly",
"responses": []
},
{
"id": "204765",
"date": "20 Oct 2023",
"name": "Werner Albrich",
"expertise": [
"Reviewer Expertise Antibiotic resistance",
"antibiotic therapy",
"respiratory infections"
],
"suggestion": "Not Approved",
"report": "Not Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe paper by Siregar et al. has been interesting to read. In a retrospective cohort study design they investigated risk factors for the presence of an extended-spectrum beta lactamase (ESBL) phenotype in patients with Escherichia coli-bacteremia and also for mortality-associated factors in those being diagnosed with ESBL-producing E. coli bacteremia.\nWhile the emergence of these pathogens is of great clinical importance and identifying high-risk patients is of value for guiding empiric treatment decisions, the present study does not add any novel findings. However, the study underlines generally known risk factors in the specific population of two Indonesian referral hospitals with a high proportion of cancer patients where the baseline prevalence of ESBL-producing E. coli is deemed to be high. Furthermore, the citation of supporting data in the introduction and discussion part is of scientific value. Data analysis and statistical methods are adequately chosen.\nHowever, there are several issues besides the limitations arising from the nature of the study design that are not congruously addressed:\nThe description of the study population is unsatisfactory, especially as the definition of some variables lacks preciseness, e.g. time intervals for the qualification of a “history of hospitalisation” or “prior antibiotic therapy” (time frame, what type of cephalosporin), time frame considered for the variable “mortality” (e.g. 30-day-mortality? In-hospital mortality?), indication for blood cultures (only medically indicated? Fever? All patients hospitalized?), clinical interpretation of positive blood cultures, definition of “sepsis” (unclear criteria), details of “empirical therapy” (which agents were used in the two groups? Time to adequate therapy?), details of “medical devices” (how long in place). Importantly, there is a complete lack of description of the definitive therapy, which is likely most relevant. Differentiation of the types of ESBLs? Were there carbapenemase producers as well? This should be detailed more clearly in the Methods section.\n\nThe author describe hypothesis testing, but they do not provide their hypothesis.\n\nThe number of cases seems low in light of the high local prevalence of ESBL carriage. Longer interval for data collection would have increased statistical power.\n\nThe introduction fails to illustrate the scientific and clinical relevance of the findings and how they may translate into scientific advancement or every day clinical decision making.\n\nThe clinical relevance of the findings can be put into question. In our view, especially in severely ill patients, it is questionable if the additional information of specific risk factors for ESBL E. coli compared to non-ESBL E. coli BSI will have a significant impact on the choice of an empirical therapy in an epidemiological setting with an ESBL-prevalence of 60-85%.\n\nThe discussion elaborates rather lengthy on topics that are only tangentially touched by the data presented (e.g. ESBL-bacteremia in children) or already known (e.g. gastrointestinal tract as a major source of E. coli).\n\nA shorter and concise discussion embedding their data into existing literature would be desirable.\n\nMost importantly, it would be of interest if the mortality rates differed between ESBL- and Non-ESBL E. coli, or if the choice of empirical and definite antibiotic therapy is associated with different mortality rates. For this it is crucial to analyse risk factors in their non-ESBL group in the same manner to see if they differ for ESBL- and non-ESBL E. coli bacteremia. Identification of sepsis as a risk factor for mortality on the other hand is a well-known fact.\nMinor comments:\nThere were a few typographical errors, which are not detailed here.\nUnfortunately, the study in its current form does not provide novel findings to justify a indexing. We rather think that these findings are relevant for local caregivers and are better suitable for local dissemination.\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? Partly\n\nAre the conclusions drawn adequately supported by the results? No",
"responses": []
}
] | 1
|
https://f1000research.com/articles/11-1449
|
https://f1000research.com/articles/11-1448/v1
|
07 Dec 22
|
{
"type": "Case Report",
"title": "Case Report: Euglycemic diabetic ketoacidosis led by empagliflozin: A case report and literature review",
"authors": [
"Irina Balan",
"V Lakshmi N Priyanka Ganapathiraju",
"Sudha Dirisanala",
"Shafaq Taj",
"Pratikkumar Vekaria",
"V Lakshmi N Priyanka Ganapathiraju",
"Sudha Dirisanala",
"Shafaq Taj",
"Pratikkumar Vekaria"
],
"abstract": "Introduction: Sodium-glucose co-transporter 2 inhibitors (SGLT2i) are a new class of medications that have been approved for the treatment of heart failure (HF) in patients with and without type-2 diabetes mellitus. It is important to be aware of the likely side effects of SGLT2i for their optimal use and enhanced patient safety. One such rare but potential side effect is the development of euglycemic diabetic ketoacidosis (EDKA). Objective: We present a case report of EDKA, in a patient who was started on empagliflozin – one of the SGLT2i – highlighting its presenting signs and symptoms, pertinent laboratory findings, differential diagnosis, treatment and outcome. To strengthen our findings and hypothesis, we conducted a literature review of other cases that used SGLT2i and found similar complications. This case report with review can help recognize the serious, potentially life-threatening complications of the new class of medication SGLT2i that has been incorporated into the current practice, and also help to take appropriate steps to mitigate its adverse effects and improve overall health outcomes in our patients. Conclusions: SGLT2i are increasingly used because of their favorable effects on mortality in the chronic HF patients along with its benefits of weight loss and blood pressure reduction. A potential underdiagnosed adverse effect of SGLT2i use is diabetic ketoacidosis in a setting of normal blood glucose levels. Thus, it is reasonable to be cognizant of its side effects to prevent any untoward events in a timely manner.",
"keywords": [
"sodium-glucose co-transporter 2 inhibitor (SGLT2i)",
"empagliflozin",
"euglycemic diabetic ketoacidosis (EDKA)",
"chronic heart failure",
"diabetes mellitus"
],
"content": "Introduction\n\nBased on the most recent Center for Disease Control and Prevention (CDC) report, approximately 6.2 million adults in the United States have chronic heart failure (HF) with a mortality rate of 13.4%, regardless of the major cardioprotective effects of the notable pharmacological HF agents (https://www.cdc.gov/heartdisease).1 In May 2020, the US Food and Drug Administration (FDA) implemented a new approach by approving the use of sodium-glucose co-transporter 2 inhibitors (SGLT2i) in the treatment of HF with reduced ejection fraction in patients with or without type-2 diabetes mellitus (T2DM) (https://www.fda.gov/news-events).\n\nMultiple studies revealed the complex direct cardiac effects of SGLT2i, including anti-inflammatory properties by reducing the macrophage infiltration, the activity of C-reactive protein (CRP) or enhancing the messenger RNA of interleukin 102,3; glycosuria that changes the whole-body metabolism to mobilization and use of lipids, and stimulates the hepatic gluconeogenesis4; involvement in the ionic cardiomyocytes metabolism by direct inhibition of Na+/H+ exchange and increasing Ca2+ reuptake and activity of sarcoplasmic endoplasmic reticulum Ca2+-ATPase (SERCA2a), therefore improving the left ventricular diastolic dysfunction.2 The cardiorenal protective effects involve the enhancement of sodium concentration in the distal macula and reduction of intraglomerular hypertension and hyperfiltration, resulting in 40–50% decrease of the estimated glomerular filtration rate (eGFR) decline.3,5\n\nConsidering the limited information currently available regarding the severe side effects of the SGLT2i, the cause of numerous cases of diabetic ketoacidosis (DKA) could not be directly related to this relatively new pharmacological group.6 Certainly, not every patient using SGLT2i has a high risk of developing euglycemic DKA (EDKA), although the potential mechanisms comprise the increase of glycosuria and reduction of serum insulin, subsequently inducing the lipolysis and production of free fatty acids that could be converted in ketones by the hepatic β-oxidation.6 The precipitating factors induced by decreased carbohydrates intake, considered a euglycemic status, are increased insulin/glucagon ratio and insulinopenia that would activate the ketogenesis.7\n\nWe have also provided data on previously published case reports/case series, management, and overall outcomes to provide a comprehensive view of potential side effects (EDKA) and their management. We searched the material for this case report and literature review through Google Scholar and PubMed.\n\nEDKA is frequently a delayed diagnosis due to relatively lower glycaemia; however, ketoacidosis could become life-threatening in case of prolonged fasting, bariatric surgery, gastroparesis or hepatopathy.8 The incidence of EDKA has increased since the introduction of SGLT2i used in the treatment of chronic heart failure and we aim to provide a complex presentation of a series of cases, including our own case.\n\n\nCase presentation\n\nA 53-year-old Caucasian female with a past medical history of uncontrolled diabetes mellitus on long-term insulin, hypertension and chronic kidney disease stage-4 (CKD-4) presented in August 2021 with a chief complaint of dizziness followed by a fall. The patient reported that she was feeling dizzy after standing up for two to three days. The patient fell a day before coming to the hospital, but she didn’t endorse loss of consciousness or head trauma. The patient went to the urgent care for the evaluation. She stated that urgent care did some work-up, but she didn’t get any definitive answers from them, and she decided to come to the emergency room (ER) as her dizziness continued. The patient reported that she went to her nephrologist two weeks prior to coming to the ER and she was started on empagliflozin along with her chronic insulin regimen for better blood glucose control. The patient told us that her diabetes was poorly controlled.\n\nThe patient’s vitals, pertinent laboratory and imaging findings on admission were as under: Vitals: Temperature 98.7 degrees Fahrenheit (oral), pulse rate 112 per minute, blood pressure 149/64 mmHg, oxygen saturation 100% on room air.\n\nFurthermore, chest x-ray showed no evidence of acute cardiopulmonary abnormalities. The patient had sinus tachycardia without any acute ST segment/T-wave changes on the electrocardiogram (ECG). The patient was diagnosed with euglycemic diabetic ketoacidosis (EDKA) in the setting of recently started empagliflozin, urinary tract infection (UTI) and acute kidney injury. The patient was also in sepsis on admission, but lactic acid was within normal limits. Alcohol and starvation related anion gap (AG) metabolic acidosis were ruled out given no history of alcohol use and no recent significant changes in diet or body weight. Blood and urine cultures were collected in the ER. Further details regarding the patient’s laboratory findings and progress are presented in Table 1. The patient was given intravenous (IV) fluid bolus and started on ceftriaxone in the ER. She was initially admitted to the medical ward, but later she was transferred to the intensive care unit (ICU) and was started on IV insulin drip along with bicarbonate-dextrose 5% in water (D5W) drip.\n\nGradually, the patient’s bicarbonate levels improved, and the anionic gap was closed. The patient also improved clinically, and she was able to tolerate an oral diet. She was transitioned to basal plus bolus insulin regimen from the insulin drip. Blood and urine cultures came back unremarkable after 72 hours. The patient’s renal function also improved, and serum creatinine was 1.55 on the day of discharge. She was advised to stop taking empagliflozin as it was the potential cause of developing UTI and EDKA in this case. She was provided follow-up with a primary care physician for further diabetes management.\n\n\nDiscussion\n\nSGLT2i are relatively new non-insulin and oral hypoglycemic agents that cause glycosuria by inhibiting the glucose reabsorption in proximal renal tubules.9 In addition to better glycemic control and cardioprotective effects, there are other metabolic benefits, including improvement of lipids, insulin resistance, nonalcoholic steatohepatitis, and weight associated with the use of SGLT2i.6\n\nSGLT2i has been a drug of choice for patients with comorbidities like hypertension and obesity because of their favorable effects on the blood pressure and weight/body mass index.10 There is an average 1.5–2 kg weight loss compared to placebo, and a clinically notable reduction by approximately 2.5–5.0 mm Hg and 1–2 mm Hg for systolic and diastolic blood pressure respectively.11,12 The EMPA-REG OUTCOME (Empagliflozin Cardiovascular Outcome Event Trial in Type 2 diabetes Mellitus Patients) study showed a 38% relative risk reduction in deaths from cardiovascular (CV) causes in patients receiving empagliflozin in comparison to placebo, and a CANVAS-R (CANagliflozin cardiovascular Assessment Study-Renal) program demonstrated that canagliflozin had a lower risk of cardiovascular events than those who received placebo.10 The data from randomized controlled CV outcome trials with all SGLT2i showed multiple cardiovascular benefits like a 30% reduction in hospital admission for HF and a reduction in deaths due to heart failure or arrhythmia.13 The Dapagliflozin and Prevention of Adverse Outcomes in Heart Failure (DAPA- HF) trial showed a 26% reduction in worsening of heart failure with reduced ejection fraction (HFrEF) or cardiovascular death, with a median duration of follow-up of 1.5 years.12 The studies using large databases from multiple countries have reassured us that these benefits can be reproduced in routine clinical practice.12\n\nSGLT2i shows glycemic control efficacy and a meta-analysis of 58 studies with eight different SGLT2i showed a reduction in mean HbA1c by 0.61% when used as add-on therapy compared to placebo.14 All SGLT2i can be initiated if the estimated glomerular filtration rate (eGFR) is more than 60 mL per min per 1.73 m2 and should be reviewed if the eGFR is less than 45 mL per min per 1.73 m2 because of a diminished glucose lowering effect.12 Renal disorders are common in T2DM, with approximately 50% of patients developing some degree of renal impairment and an increasing prevalence of both conditions over time.13 Recent trials with SGLT2i suggested their potential to reduce the rates of end stage renal disease (ESRD) and acute kidney injury (AKI).13 Multiple renal risk markers like eGFR, urinary albumin: creatinine ratio (UACR), and serum uric acid levels have been used as risk measurements together with hard outcomes like ESRD or renal death in five major CV outcome trials, thus indicating that SGLT2i could prevent the development and/or delay the worsening of CKD in people with T2DM at any level of renal risk.13 Analyses on CV outcome trials have shown that there may be fewer AKI events in patients on SGLT2i compared to placebo.13\n\nConsidering the above-mentioned beneficial effects of SGLT2i, the use of these medications has been increasing in day-to-day practice by the providers in all clinical settings. Thus, it is reasonable to be cognizant of its side effects at the same time to prevent any untoward events in a timely manner. A potentially underdiagnosed adverse effect of SGLT2i use is diabetic ketoacidosis with a reported incidence of less than 0.2% in type 2 and 9.4% in type 1 diabetic patients.9 We collected data on 16 recently published case reports showing development of diabetes ketoacidosis with and without euglycemia in the patients started on SGLT2i.\n\nIn order to support our hypothesis, we combined our case report with an extensive literature review that can help promote evidence-based practice in the real world to improve the health of patients and community. To summarize, all case reports mentioned below in Table 2 endorse the findings of our case presentation, specifically SGLT2i– related side effects. We also tried to collect information on the duration of SGLT2i exposure before the development of EDKA and found the range of 1 dose to six years which suggests an uncertain latent time before the patient can develop EDKA. We can construe that overall management is almost the same in all case reports which include treatment with insulin drip, intravenous hydration, and cessation of SGLT2i.\n\nThe limitations of our case report and literature review include limited availability of data on laboratory parameters and pre-existing systematic conditions that could have contributed to the induction of DKA. There is limited data on the duration of drug usage in correlation with the incidence of euglycemic DKA. We only included case reports found on the PubMed database and written in English which may lead to missing case reports in other languages.\n\n\nConclusions\n\nThe use of SGLT2i has increased significantly in recent times. SGLTi are not only implicated in the treatment of diabetes, but also in the management of chronic HF, hypertension and CKD. As SGLT2i has been approved for use in these multiple disease conditions, regardless of patients’ diabetes status, it is thus imperative for the clinical providers to equip themselves with the knowledge of possible side effects of SGLT2i. EDKA has been found to be one of the significant and life-threatening adverse effects of SGLT2i use. Therefore, it is crucial for physicians to monitor patients who use this medication for signs and symptoms of DKA in the setup of normal glucose levels. This will facilitate early diagnosis, obviate hospitalizations and associated patient morbidity, and thus expedite effective patient management and their disposition.\n\n\n\n• The use of sodium-glucose co-transporter 2 inhibitors (SGLT2i) have been on the rise for the treatment of various disease conditions including chronic heart failure, blood pressure and weight loss with or without concomitant diabetes mellitus.\n\n• It is imperative to be aware of potential life-threatening complications of SGLT2i such as euglycemic diabetic ketoacidosis.\n\n• Physicians should closely monitor patients who use this class medications for signs and symptoms of diabetic ketoacidosis (DKA) in the setting of normal glucose levels to improve patient safety and health outcomes.\n\n\nConsent\n\nWritten informed consent for publication of their clinical details 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\nGenuardi MV, Mather PJ: The dawn of the four-drug era? SGLT2 inhibition in heart failure with reduced ejection fraction. Ther. Adv. Cardiovasc. Dis. 2021 Jan-Dec; 15: 175394472110026. PubMed Abstract | Publisher Full Text | Free Full Text\n\nLahnwong S, Chattipakorn SC, Chattipakorn N: Potential mechanisms responsible for cardioprotective effects of sodium–glucose co-transporter 2 inhibitors. Cardiovasc. Diabetol. 2018; 17: 101. Publisher Full Text\n\nZeng Q, Zhou Q, Liu W, et al.: Mechanisms and Perspectives of Sodium-Glucose Co-transporter 2 Inhibitors in Heart Failure. Front Cardiovasc Med. 2021 Feb 10; 8: 636152. PubMed Abstract | Publisher Full Text | Free Full Text\n\nFerrannini E, Baldi S, Frascerra S, et al.: Shift to Fatty Substrate Utilization in Response to Sodium-Glucose Cotransporter 2 Inhibition in Subjects Without Diabetes and Patients With Type 2 Diabetes. Diabetes. 2016 May; 65(5): 1190–1195. Epub 2016 Feb 9. PubMed Abstract | Publisher Full Text\n\nChambergo-Michilot D, Tauma-Arrué A, Loli-Guevara S: Effects and safety of SGLT2 inhibitors compared to placebo in patients with heart failure: A systematic review and meta-analysis. Int J Cardiol Heart Vasc. 2020 Dec 11; 32: 100690. PubMed Abstract | Publisher Full Text | Free Full Text\n\nOgawa W, Sakaguchi K: Euglycemic diabetic ketoacidosis induced by SGLT2 inhibitors: possible mechanism and contributing factors. J Diabetes Investig. 2016 Mar; 7(2): 135–138. Epub 2015 Sep 6. PubMed Abstract | Publisher Full Text | Free Full Text\n\nYu X, Zhang S, Zhang L: Newer Perspectives of Mechanisms for Euglycemic Diabetic Ketoacidosis. Int. J. Endocrinol. 2018 Oct 2; 2018: 1–8. PubMed Abstract | Publisher Full Text | Free Full Text\n\nNasa P, Chaudhary S, Shrivastava PK, et al.: Euglycemic diabetic ketoacidosis: A missed diagnosis. World J. Diabetes. 2021 May 15; 12(5): 514–523. PubMed Abstract | Publisher Full Text | Free Full Text\n\nClark A, Mohammed AS, Raut A, et al.: Prevalence and Clinical Characteristics of Adults Presenting With Sodium-Glucose Cotransporter-2 Inhibitor-Associated Diabetic Ketoacidosis at a Canadian Academic Tertiary Care Hospital. Can. J. Diabetes. 2021 Apr; 45(3): 214–219. Epub 2020 Aug 21. PubMed Abstract | Publisher Full Text\n\nGuirguis H, Beroukhim Afrahimi S, Pham C: The Use of SGLT-2 Inhibitors Coupled With a Strict Low-Carbohydrate Diet: A Set-Up for Inducing Severe Diabetic Ketoacidosis. Clin Med Insights Case Rep. 2022 Apr 8; 15: 117954762210900. PubMed Abstract | Publisher Full Text | Free Full Text\n\nPereira MJ, Eriksson JW: Emerging Role of SGLT-2 Inhibitors for the Treatment of Obesity. Drugs. 2019 Feb; 79(3): 219–230. PubMed Abstract | Publisher Full Text | Free Full Text\n\nPacker M, Anker SD, Butler J, et al.: EMPEROR-Reduced Trial Investigators. Cardiovascular and Renal Outcomes with Empagliflozin in Heart Failure. N. Engl. J. Med. 2020 Oct 8; 383(15): 1413–1424. Epub 2020 Aug 28. PubMed Abstract | Publisher Full Text\n\nGiorgino F, Vora J, Fenici P, et al.: Renoprotection with SGLT2 inhibitors in type 2 diabetes over a spectrum of cardiovascular and renal risk. Cardiovasc. Diabetol. 2020 Nov 22; 19(1): 196. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMikhail N: Place of sodium-glucose co-transporter type 2 inhibitors for treatment of type 2 diabetes. World J. Diabetes. 2014 Dec 15; 5(6): 854–859. PubMed Abstract | Publisher Full Text | Free Full Text\n\nChacko B, Whitley M, Beckmann U, et al.: Postoperative euglycaemic diabetic ketoacidosis associated with sodium-glucose cotransporter-2 inhibitors (gliflozins): a report of two cases and review of the literature. Anaesth. Intensive Care. 2018; 46(2): 215–219. Publisher Full Text\n\nDull RB, Spangler ML, Knezevich EL, et al.: Euglycemic Diabetic Ketoacidosis Associated With Sodium-Glucose Cotransporter Type 2 Inhibitors in Patients With Type 2 Diabetes Mellitus Receiving Oral Therapy. J. Pharm. Pract. 2019; 32(2): 240–243. Publisher Full Text\n\nElshimy G, Correa R: Sudden-onset Hypoglycemia Following Fluid Replacement in a Patient with Dapagliflozin-induced Diabetic Ketoacidosis Without Prior Insulin Use: Case Report. Cureus. 2019; 11(8): e5448. Published 2019 Aug 21. Publisher Full Text\n\nAdachi J, Inaba Y, Maki C: Euglycemic Diabetic Ketoacidosis with Persistent Diuresis Treated with Canagliflozin. Intern. Med. 2017; 56(2): 187–190. Publisher Full Text\n\nMiwa M, Nakajima M, Kaszynski RH, et al.: Prolonged euglycemic diabetic ketoacidosis triggered by a single dose of sodium-glucose cotransporter 2 inhibitor. BMJ Case Rep. 2020; 13(10): e235969. Published 2020 Oct 7. Publisher Full Text\n\nLee IH, Ahn DJ: Dapagliflozin-associated euglycemic diabetic ketoacidosis in a patient with type 2 diabetes mellitus: A case report. Medicine (Baltimore). 2020; 99(21): e20228. Publisher Full Text\n\nRafey MF, Butt A, Coffey B, et al.: Prolonged acidosis is a feature of SGLT2i-induced euglycaemic diabetic ketoacidosis [published online ahead of print, 2019 Sep 27]. Endocrinol. Diabetes Metab. Case Rep. 2019; 2019: 19–0087. Publisher Full Text\n\nKitahara C, Morita S, Kishimoto S, et al.: Early detection of euglycemic ketoacidosis during thoracic surgery associated with empagliflozin in a patient with type 2 diabetes: A case report. J Diabetes Investig. 2021; 12(4): 664–667. Publisher Full Text\n\nDiaz-Ramos A, Eilbert W, Marquez D: Euglycemic diabetic ketoacidosis associated with sodium-glucose cotransporter-2 inhibitor use: a case report and review of the literature. Int. J. Emerg. Med. 2019; 12(1): 27. Published 2019 Sep 5. Publisher Full Text\n\nTurner J, Begum T, Smalligan RD: Canagliflozin-Induced Diabetic Ketoacidosis: Case Report and Review of the Literature. J. Investig. Med. High Impact Case Rep. 2016; 4(3): 232470961666323. Published 2016 Aug 29. Publisher Full Text\n\nMistry S, Eschler DC: Euglycemic Diabetic Ketoacidosis Caused by SGLT2 Inhibitors and a Ketogenic Diet: A Case Series and Review of Literature. AACE Clin Case Rep. 2020; 7(1): 17–19. Published 2020 Dec 28. Publisher Full Text\n\nSethi SM, Vohra M, Ali SA: EUGLYCEMIC DIABETIC KETOACIDOSIS (EDKA) IN A PATIENT RECEIVING DAPAGLIFLOZIN. Acta Endocrinol (Buchar). 2021; 17(2): 266–269. Publisher Full Text"
}
|
[
{
"id": "158062",
"date": "03 Jan 2023",
"name": "Syed Nazeer Mahmood",
"expertise": [
"Reviewer Expertise Internal 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\nSGLT2 inhibitors are a class of medication that can be used to treat heart failure in people with and without diabetes. It is important to be aware of the possible side effects of these medications, including the rare possibility of developing euglycemic diabetic ketoacidosis (EDKA).\nThe authors report a case where the patient developed EDKA while on empagliflozin, a type of SGLT2 inhibitor. The report includes details on the signs and symptoms of the condition, relevant laboratory test results, and the treatment and outcome of the case with sufficient detail in a manner in which the teaching points can be extrapolated to general practice. The report also includes a review of other cases in the literature where SGLT2 inhibitors presented with similar complications. The case highlights a side effect of this class of medication that healthcare practitioners must be aware of.\nSuggested changes pertinent to each section:\nAbstract:\nWould remove \"reasonable\" from the last sentence in the conclusion section. Can replace it with \"important\".\n\nIntroduction:\n\nChange “converted in ketones” to “converted into ketones”.\n\nThe current mechanism suggested as per the quoted reference – 7 is: Decreased carbohydrate intake causes insulinopenia and increased glucagon. Increased glucagon/insulin ratio further promotes lipolysis and ketogenesis. Please change the ratio to reflect increased glucagon/insulin instead of “insulin/glucagon”.\n\nCase Presentation:\n\nPlease add units for all the lab values in the table as well.\n\nAs there are a few differentials here, it may be beneficial to edit the case report to reflect what was considered first and how things were ruled out before reporting that EDKA was the eventual diagnosis.\n\nDiscussion:\n\nPlease maintain consistency with regarding acronyms for the trials quoted, if you were to mention the acronym for each trial in parenthesis, please do so for the EMPA-REG OUTCOME and CANVAS-R trials as well.\n\nIt may be useful to mention that all of these case reports have been in patients with a known history of DM, given that this class of drug is now approved to be used in patients without a history of DM for cardioprotective benefits in HF, it might be beneficial to know that this side effect is likely to occur in the DM population.\n\nConclusions:\nWould change \"implicated\" to \"SGLTis are not only used in … \".\n\nThe conclusion goes on to emphasize the threat of EDKA although data in the non-diabetic population is limited. It may be useful to keep that open-ended and the need for further studies in that particular population.\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": "240271",
"date": "07 Feb 2024",
"name": "Fateen Ata",
"expertise": [
"Reviewer Expertise Endocrine system"
],
"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 claims in the introduction that the incidence of Euglycemic Diabetic Ketoacidosis (EDKA) has risen with the use of SGLT2 inhibitors. This assertion, while technically accurate due to the expanded use of these drugs, lacks the necessary context to fully understand the implications. It is critical to note that despite the perceived increase, EDKA remains an exceedingly rare adverse effect. The apparent increase in the rare side effect is due to the increase in the prescription of the drug for obvious reasons. The introduction would benefit from highlighting the relative rarity of EDKA and including well-established data on the incidence of Diabetic Ketoacidosis (DKA), including EuDKA and HDKA, in the context of SGLT2 inhibitor use, along with a clinical perspective on these findings.\nFurthermore, attributing DKA directly to SGLT2 inhibitor use without acknowledging the complexity of causation oversimplifies the issue. The terms used in the manuscript to describe the relationship between SGLT2 inhibitors and DKA are exaggerated and must be revised to more accurately reflect the nuanced nature of drug-related adverse effects. For example, the statement \"EDKA has been found to be one of the significant and life-threatening adverse effects of SGLT2i use\" could mislead readers regarding the actual clinical significance of EDKA in the context of SGLT2 inhibitor therapy. A review of recent, larger cohort studies would illustrate that the incidence of EDKA, while non-negligible, is relatively low. Acknowledging the risk without overstating its significance is crucial to maintaining a balanced view of the drug's overall efficacy and safety profile.\nTo improve the manuscript's accuracy and objectivity, I recommend a major revision that addresses these concerns. Specifically, the authors should revise the language to present a more balanced and data-driven discussion of the risks associated with SGLT2 inhibitors, including a clearer representation of the incidence and clinical context of EDKA. Adjusting the tone and content of the article to incorporate these suggestions will greatly enhance its value to the scientific community and clinical practice.\n\nIs the background of the case’s history and progression described in sufficient detail? No\n\nAre enough details provided of any physical examination and diagnostic tests, treatment given and outcomes? Partly\n\nIs sufficient discussion included of the importance of the findings and their relevance to future understanding of disease processes, diagnosis or treatment? Partly\n\nIs the case presented with sufficient detail to be useful for other practitioners? Partly",
"responses": []
}
] | 1
|
https://f1000research.com/articles/11-1448
|
https://f1000research.com/articles/11-325/v1
|
17 Mar 22
|
{
"type": "Research Article",
"title": "Electronic signature and attestation in conveyancing practice: A Malaysian legal perspective",
"authors": [
"Hua Siong Wong",
"Mohd Munzil Muhamad",
"Mohd Munzil Muhamad"
],
"abstract": "Background The Corona Virus Disease 2019 (COVID-19) pandemic brought about an unprecedented disruption to global business activities. Physical face-to-face activities must be restricted due to movement control order (MCO). The clients are required to sign the documents physically in the presence of the solicitor who must subsequently attest to the signature of the clients. The issue arises whether electronic signature (e-signature) and attestation are permissible under the laws of Malaysia. The aim of this research was to study the legality of e-signature and attestation in conveyancing practice in Malaysia and subsequently to propose recommendations to overcome these issues.\n\nMethods\n\nThis is qualitative study and not an empirical study. The data was collected by library-based research from various primary and secondary data sources, including case law in Malaysia, written statutes, publication of journal and article.\n\nResults The Digital Signatures Act 1997 (DSA) and the Electronic Commerce Act 2006 (ECA) have legalised e-signatures. The DSA is the law that governs the digital signatures in Malaysia. ECA has listed a few documents which are not legally accepted if signed electronically, namely Power of Attorney, the Wills and codicils, the Trusts, and negotiable instruments. However, with regards to the issue of attestation of these documents, there are no clear laws which govern the attestation. The legal issue arises when the lawyers who have attested these documents are liable to be called as witness under the Evidence Act 1950 to testify their signature if these documents are tendered as evidence in any court proceedings.\n\nConclusion Thus, it is suggested that there is a need for unique legal framework for e-signature and attestation in Malaysia due to the lack of specific laws which govern the issues of electronic signature and attestation.",
"keywords": [
"Attestation",
"Conveyancing Practice",
"Electronic Signature",
"Malaysia."
],
"content": "Introduction\n\nThe Corona Virus Disease 2019 (COVID-19) pandemic brought about an unprecedented disruption to the global business activities, including but not limited to the importing and exporting, franchising, joint venture, service activities, i.e., hotel, legal service, retails etc. Due to the movement control order (MCO) and strict standards of procedure (SOP), most of the physical face-to-face activities must be restricted in order to prevent physical contact. In legal line service, especially law firms which handle the property transactions and conveyancing practice, involve a lot of documentation that will require the clients’ signature and subsequently to be attested by the solicitors. However, due to the MCO and that the law firms are not considered as “essential service”, the law firms in West Malaysia are required to temporarily close. Thus, it has been suggested by many to substitute the physical signatures with electronic signatures (e-signatures) followed by document attestation to be done by video conference calls. In practice, some judges would accept affidavits which have yet to be affirmed subject to solicitors’ undertaking to duly affirm them after the MCO. This scenario can be seen in the case of SS Precast Sdn. Bhd. Vs. Serba Dinamik Group Bhd. & Ors [2020] MLJU 400. Due to the MCO, the counsel was not able to affirm the affidavit before a Commissioner for Oaths. In this case, the learned High Court judge Datuk Wong Kian Kheong emphasized that after the MCO, any affirmation of the documents still requires the signatures of the Commissioner for Oaths with wet-ink.\n\nAlthough e-signature is not a new technology in Malaysia, the issue remains whether e-signature and attestation of the legal documents via video conferencing platform are permissible under the Malaysian laws.\n\nThe aim of this research is to study the legality of e-signature and attestation in conveyancing practice in Malaysia. Secondly, the author will identify the legal issues relating to e-signature and attestation and thirdly, the author will propose recommendations to overcome these issues.\n\n\nMethods\n\nThis study is a qualitative study and adopted doctrinal research methodology. The appropriate data were collected from the library, relevant databases, and other archives. The contents of the current statutory provisions, case law and guidelines on the issues of e-signature and attestation of the documents in the legal practice were analyzed, especially during the Covid-19 pandemic.\n\nThis study has explored both primary and secondary data. The primary data included the current statutes, case law, rules and guidelines on e-signature and attestation of the document (See Underlying data) (Hua Siong, 2021). The relevant articles and journal publication and reference textbooks were categorized as secondary data. Data from library-based research was done in various libraries from local government and private universities. The interviews were performed among the selected respondents.\n\nMeanwhile, this study used content analysis as the main method of data analysis. This method provided an overview of the study and relevant evidence for the facts.\n\n\nResults/Discussion\n\nGenerally, a signature is proof that the signatory has read and agreed to be bound by the contents of the document. Additionally, it indicates that the signatory has specifically consented to the contents of the documents and wanted it to have a legal effect.\n\nThe thumb-print has also been defined as ‘signature’ under the Section 3 of the Interpretation Acts of 1948 and 1967 (Consolidated and Revised 1989) (Act 388) in Malaysia. Under section 210(2) of the National Land Code 1956, a thumb-print on the instrument of dealings is considered legal and valid. However, this type of signature may not serve as conclusive evidence as it may be challenged by other factors, namely duress, undue influence, fraud, forgery etc.\n\nIn Malaysia, legislations governing electronic signature include Digital Signature Act 1997 (DSA), Electronic Commerce Act 2006 (ECA) and Electronic Government Activities Act 2007 (EGAA). E-signatures in Malaysia are governed by the Electronic Commerce Act (ECA) 2006. However, for digital signatures, the relevant legislation would be the Digital Signature Act (DSA) 1997.\n\nIt is important to understand the differences between digital signatures and e-signatures. Generally, e-signature is not so complex when compared to a digital signature which requires a lot of procedures, such as authentication of public and private keys, digital certificate etc. Under sections 22-26 of DSA, these signatures require licenced certification authorities, and such activities can only be performed as specified in the licence. Both signatures are legally recognized in Malaysia by DSA and ECA. Furthermore, for electronic commercial transactions both signatures can be used under the ECA. If digital signature is chosen, then provisions under DSA will be applied. Section 9(1) and (2) of the ECA also recognizes the contracts which have been finalised by the parties in the contracts and were executed electronically, however such contracts and agreements are subject to the Stamp Act. Under the Malaysia Evidence Act 1950, the court accepts and admits any agreements which are signed electronically provided that these documents passed the rules of admissibility of evidence. Under the section 90A of the Evidence Act 1950, it states that the court shall admit the document produced by a computer, or a statement contained in such document as evidence if the document was produced by the computer during its ordinary use, whether the person tendering the same is the maker of such document or statement (Radhakrishna, 2012).\n\nMeanwhile, the usage of Personal Identification Number (PIN) is in fact permissible under the Prescription of Electronic Signature Order 2010 issued pursuant to the Electronic Government Activities Act 2007. Thus, there is no legal issue about the usage of PIN as electronic signature.\n\nAccording to Nguyen (2013), a digital signature can be defined as one form of e-signature, that is highly safe and widely used. Based on Huang, Chen, and Hoa (2010), digital certificates are an electronic file that can be used as an identity card to verify the identity of a party on the Internet. In order for digital signature to be functionable, it requires a digital certificate which is also known as private key for verification purpose (Kumar, 2019). This private key is only known by the person who is authorised to access the document. The public key held by the owner and creator of the document must correspond with the private key for the purpose of determination of the legitimacy of this digital signature and to track any changes made in the document. Figure 1 briefly explains the process of digital signature.\n\nSource: https://signx.wondershare.com/knowledge/digital-signature-example.html.\n\nDigital signature is a good authentication tool as it can provide privacy and high integrity (Jason 2014). Nevertheless, there is still a possibility for a third party to create a false digital signature by using false identity. For example, a hacker can create fake encryption keys and register the public key under the name of individual A. The hacker can use the fake private key of individual A to encrypt a message and sent it to individual B. Upon receiving the message, individual B will think that it is a genuine message from individual A. However, as the content of the message is not correct, it will mislead individual B, or it may even contain harmful executable viral software that could hack the receiver’s computer or system. However, in this paper, we will focus on the issue of e-signature.\n\nMalaysia’s Electronic Commerce Act 2006 has adopted the United Nations Commission on International Trade Law (UNCITRAL) Model of Electronic e-signature and as such e-signatures are accepted and recognized worldwide. This practice has become more popular among corporate transactions, including the commercial agreements between corporate entities and due diligence documents for corporate proposals which involve transactions of more than million US dollars. However, the security issue of e-signatures is always a priority to e-signature users.\n\nPart III of the ECA recognizes that e-signatures are legally enforceable provided that the following requirements are fulfilled including but not limited to (I) satisfying the definition of an e-signature in ECA; (II) the signature being logically associated with the electronic document; (III) to be able to identify the signature of the owner and obtain the approval of the information in the document; (IV) the signature to be under control of the owner; and (V) any changes in the signature or the associated documents to be detectable.\n\nThe ECA has listed few documents which cannot use e-signature in the same effect due to the security and trust issues, these documents include but are not limited to Power of Attorney, the creation of Wills and Codicils, the creation of Trusts, and negotiable instruments, particularly in banking documents. Another reason that these documents in specific Wills and Codicils, Trusts are not allowed to use e-signatures is to prevent fraudulent activities from the third parties as these are based on the intention of the makers.\n\nMeanwhile, some commercial documents, namely company’s annual accounts, share transfer instruments, and documents from the court may require formal attestation from the Commissioner for Oaths or notarization by a Notary Public or a seal to be affixed under any other laws, i.e., Companies Act 2016, as such e-signatures will not be suitable.\n\nTo date, there were not many cases that deliberated on the application of the ECA. However, the recent case of Yam Kong Seng & Anor v Yee Weng Kai [2014] 4 MLRA 316 does illustrate the point above that it is not necessarily a very difficult threshold to meet in order to identify the signatory. In that case, the court has agreed to accept even a simple short message service (SMS) to be considered as fulfillment of the legal requirement for a signature. It is important to note that s.66(4) of the Malaysia Companies Act 2016 (“CA 2016”) states that it is not compulsory to use common seal for a company’s document as it can also be signed by an authorized officer. If the authorized officer can sign on behalf of the company, it is safe to argue that the officer’s e-signature may also be accepted provided the requirements under the ECA are fulfilled.\n\nIt is important to note that the reliability of a document, for example trade commercial agreements which use an e-signature, could still be challenged in court. This is because the e-signature is just a symbol affixed to an electronic document, it is impossible to track the changes made to the document at the time of signature. At the moment, there aren’t any tools that can verify the identity of the person, or whether the document was signed voluntarily under duress conditions. To reduce the possibility of authentication being raised, all these practical difficulties should be addressed.\n\nDue to restriction on in person meetings, it is important to understand whether a conveyancing solicitor can attest the signing of a document via video conferencing platform, namely Zoom or Google Meet. In regard to this issue, the Malaysian law pertaining to the attestation of the documents or witnessing a signature via video conferencing is still very uncertain and silent to whether they legally recognize this kind of process. As Malaysian courts have started to transition to online hearings gradually and steadily (Jeyakuhan, 2020), Bar Council Malaysia has issued their latest Circular No. 222/2021 to clarify the issue of attestation of the signing of the documents via video conferencing platform. Pursuant to this Circular, the solicitors are advised to seek a third-party legal advice before proceedings. However, the Circular is not clear as to whether the solicitors are permitted to attest the signing of the documents via video conferencing platform. The main purpose for the presence of a solicitor is to witness and attest signing of a document is to prove the identity of the signature and to make sure that the document is getting signed voluntarily and not under the duress. Section 15 of the Malaysia Contracts Act 1950 defined the meaning of coercion. In the case of Kanhaya Lal v National Bank of India Ltd (1913) 40 Ind App 56 (PC) 83, the Privy Council explained that in section 15, the term “coercion” refers to an illegal conduct committed “with the goal of inducing the person to enter into an agreement.” Thus, prior to the existence of the contract, the parties must give consent to be bound by the promise and this consent must, however, be obtained freely without any pressure from the other party. Meanwhile, if the solicitor attests a signing ceremony via video conferencing, they would not be able to confirm that the signatory is not under any duress from the third party who might be present in the room but not visible on the video conference call.\n\nThe next issue is understanding the definition of “in the presence of”’ and whether it/should be given a strict or wide interpretation. Unfortunately, the laws in Malaysia are still unclear about this definition. There is an issue of whether watching someone signing a document via video conferencing would amount to “signing in the presence of this person” although both the signatory and the person who witnessed the signing were not in the same room. Besides that, it is also difficult for the witness (Solicitor) to confirm that the document that is subsequently sent to him for attestation is the same document he saw on the screen.\n\n\nConclusions\n\nIn light of Covid-19 outbreak and social distancing, legislators will likely need to review and amend existing laws to cater for current situations during the pandemic. In essence, e-signatures can work as substitute if face-to-face meetings are not practicable. However, we need to be very caution about the usage of e-signatures to avoid any misuse of its convenience. Attestation via video conferencing needs to be addressed clearly by taking into consideration the meaning of ‘in the presence of’. In order to prevent any fraudulent activities, Malaysia has also started the biometric system, especially in the dealing with land trades for collection of original land title which takes place at the relevant land office/registry. However, it needs to be considered that it is strongly advisable to have the documents to be signed physically to avoid any risk of dispute about the authenticity of the execution of the documents.\n\nIn Malaysia, it has become a practice that all the government departments for example land office and stamping office will only accept relevant documents to be signed in black fountain wet-ink. These departments do not accept any electronic e-signatures. This practice is to avoid any fraudulent transactions as the land office, including the Registrar of Titles, has a duty of care toward proprietors of the land to safeguard their interest, as indicated by cases Pendaftar Hakmilik Negeri Selangor & Ors v Shaifulizam Mohd Saleh & Anor And Another Appeal [2020] 5 CLJ 595 and Pendaftar Hakmilik Negeri Selangor v Caesius Development Sdn Bhd & Ors And Another Appeal [2020] 3 CLJ 327.\n\nIn essence, when we are not able to meet in person, e-signatures are a viable solution as Malaysian laws recognize e-signatures. However, we need to take note of the safety factors surrounding e-signatures discussed in this article. Perhaps it can be beneficial for the current ECA to be amended to allow conveyancing solicitors to attest the signing of a document via video conferencing platform. Parties are thus advised to seek independent legal advice from those who are experts in this area of law to protect their rights.\n\n\nData availability\n\nFigshare: Electronic signature and attestation in conveyancing practice: A Malaysian legal perspective https://figshare.com/articles/dataset/Cases_zip/16530738 (Hua Siong, 2021).\n\nThis project contains the following underlying data:\n\nCase 1-5. The case studies and analyses mentioned in this study.\n\nData are available under the terms of the Creative Commons Zero “No rights reserved” data waiver Attribution 4.0 International (CC BY 4.0)\n\n\nAuthor contributions\n\nConceptualization, Data Curation, Funding Acquisition, Project Administration, Writing – Original Draft Preparation, by Wong Hua Siong. Formal Analysis, Investigation, Methodology, and Resources, Writing – Review & Editing by Wong Hua Siong & Mohd. Munzil Muhamad. Supervision by Mohd. Munzil Muhamad.\n\n\nList of statutes\n\n[1] Bar Council Malaysia Circular No 222/2021.\n\n[2] Companies Act 2016.\n\n[3] Contracts Act 1950.\n\n[4] Digital Signature Act 1997.\n\n[5] Electronic Commerce Act 2006.\n\n[6] Electronic Government Activities Act 2007.\n\n[7] The Evidence Act 1950.\n\n[8] The Malaysia Companies Act 2016.",
"appendix": "Acknowledgements\n\nThe author is also grateful to Multimedia University, Malaysia in funding the fee for the Conference.\n\n\nReferences\n\nAndress J: The Basics of Information Security (Second Edition) Understanding the Fundamentals of Infosec in Theory and Practice 2014.2014. Pages 69–88\n\nHua Siong W: Cases.zip. figshare. Dataset. 2021. Publisher Full Text\n\nHuang Z, Chen Q, Hao Y: Certificate-based blind signatures from bilinear pairings. The 2nd International Conference on Information Science and Engineering. 2010; pp. 1903–1906. Publisher Full Text\n\nJeyakuhan SKJ: 2020. Digital wills and assets: Moving on with the times. Nov 1, 2020. Reference Source\n\nJohansson T, Nguyen PQ: Advances in Cryptology - EUROCRYPT 2013, 32nd Annual International Conference on the Theory and Applications of Cryptographic Techniques, Athens, Greece, May 26-30, 2013. Proceedings. Lecture Notes in Computer Science 7881, Springer. 2013. 978-3-642-38347-2.\n\nKanhaya Lal v National Bank of India Ltd: 40 Ind App 56 (PC) 83.1913.\n\nKumar M, Chand S: ESKI-IBE: Efficient and secure key issuing identity-based encryption with cloud privacy centers. Multimed. Tools Appl. 2019; 78: 19753–19786. Publisher Full Text\n\nPendaftar Hakmilik Negeri Selangor v Caesius Development Sdn Bhd & Ors And Another Appeal: 3 CLJ 327.2020.\n\nPendaftar Hakmilik Negeri Selangor & Ors v Shaifulizam Mohd Saleh & Anor And Another Appeal: 5 CLJ 595.2020.\n\nRadhakrishna G: Digital evidence in Malaysia. Digital Evidence & Elec. Signature L. Rev. 2012; 9: 31.\n\nSeng YK, Kai AvYW: 4 MLRA 316.2014.\n\nSrivastava A: Electronic signatures: A brief review of the literature. Spencer B, Fox MS, Du W, et al., editors. The New e-Commerce: Innovations for Conquering Current Barriers, Obstacles and limitation to Conducting Successful Business on the Internet: Proceedings of the Eighth International Conference on Electronic Commerce (ICEC'06). Association for Computing Machinery (ACM); 2006; (pp. 605–609). Publisher Full Text\n\nSS Precast Sdn. Bhd. v Serba Dinamik Group Bhd. & Ors: MLJU 400.2020."
}
|
[
{
"id": "130205",
"date": "28 Apr 2022",
"name": "Rod Thomas",
"expertise": [
"Reviewer Expertise Real property",
"sale of land",
"high density living",
"property law",
"equity",
"art law"
],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe authors are to be commended for raising a practical issue which needs resolution in the “Time of Covid”. I take this to be the need to satisfactorily prove the witnessing of conveyancing documents when, under covid conditions, person-to-person meetings are not permissible.\nThe article opens with a broad discussion of legal issues related to signatures and attestation. The authors recognise that, in essence, a signature is some physical “affixing” to a document showing an intention to be bound. They acknowledge that, at law, a signature may be an inked thumb print, an “e-signature” or even a “digital signature”. They also record the purpose of the Digital Signatures Act 1997 (DSA) and the Electronic Commerce Act 2006 (ECA) which, in combination, may enable a “digital” signature to be accepted for evidential purposes, providing certain statutory protocols are adhered to. The article then extends on to mention to other issues in practice, such as some government agencies only accepting signatures made in “black fountain wet-ink”. The authors reflect that, welcome as the provisions in the DSA and ECA are, those provisions do not adequately extend to situations where execution must be undertaken before a witness who is not physically present under covid conditions.\nThis last point I take to be the central issue that the article ultimately seeks to address, although this is not clear because of the preceding, rather general discussion. The question then posed, appears to be whether execution of conveyancing documents can be understood to be “before” a witness who is only available by an electronic link. The conclusion in the abstract is representative of the broad ranging discussion that has occurred. It reads as follows.\n“Thus, it is suggested that there is a need for a unique legal framework for e-signature and attestation in Malaysia due to the lack of specific laws which govern the issue of electronic signature and attestation”.\nIt seems to me that the article would have benefited from isolating out, and then starting with the key issue to be addressed. As stated, I take this to be the present need for conveyancing documents to be signed before witnesses who are physically present. The material presented could then be better positioned to discuss possible solutions. Mention of the requirements of the DSA and ECA do have their place, but only in the sense of identifying missed opportunities.\nBy the time I got to the conclusion, I was made aware of the problem, but was unclear on the solution. On the one hand the authors seem to suggest “e-signatures are a viable solution”, notwithstanding their recognition that for e-signatures, “safety factors” remain a concern. At the same time they suggest the ECA be “amended to allow conveyancing solicitors to attest the signing of a document via video conferencing platform[s]”.\nIn essence, I was left a little uncertain whether the purpose of the article was to identify all legal issues related to electronic signatures, or to discuss possible solutions for signatures to conveyancing documents that must be signed before a witness. The two topics are interrelated, but different.\nThe authors are to be commended for taking the first step into the arena. We all benefit from a discussion such as this.\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nNot applicable\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Partly",
"responses": [
{
"c_id": "8550",
"date": "21 Jul 2022",
"name": "Wong Hua Siong",
"role": "Author Response",
"response": "Thank you for the feedback and will incorporate the comments and improve the contents accordingly."
}
]
},
{
"id": "139343",
"date": "20 Jun 2022",
"name": "Noraziah Abu Bakar",
"expertise": [
"Reviewer Expertise Land Law and Conveyancing Practice in Malaysia."
],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe paper is an excellent and essential addition to the literature. However, the discussion on the attestation of instrument of dealings prescribed under the conveyancing practices in Malaysia is regulated under the National Land Code (Revised 2020) (Act 828) where it is strictly regulated by Act 828 that attestation by lawyers shall comply with Section 211 and 5th Schedule of the Act. Thus, when execution of the instrument is dealing is done by a natural person, a lawyer shall attest to the signature of the parties and sign the attestation clause provided in the instrument. The attestation indicates that the execution (signing) is done in the presence of the lawyer. The conclusions are well written but it is silent on the impact of e-signature under the abovementioned provisions of Act 828.\n\nIs the work 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/11-325
|
https://f1000research.com/articles/11-1213/v1
|
25 Oct 22
|
{
"type": "Systematic Review",
"title": "Effects of evidence-based clinical practice guidelines for breast cancer in health care quality improvements. A second systematic review.",
"authors": [
"Anggie Ramírez-Morera",
"Mario Tristán",
"Jordan Salazar-Vargas",
"Ana Leonor Rivera-Chavarría",
"Mario Tristán",
"Jordan Salazar-Vargas",
"Ana Leonor Rivera-Chavarría"
],
"abstract": "Background: Traditionally, EB-CPGs have been believed to mainly improve the quality and consistency of health care, but this claim must be conclusively proven. We used the Donabedian three-dimensional model (structure, process, and patient outcomes) to assess improvements in the quality of medical care derived from implementing EB-CPGs. This study corresponds to the second systematic review carried out as a series of studies on different clinical issues that aim to evaluate the effectiveness of the application of the EB-CPG for improving the quality of care. Methods: We followed the methods described by the Cochrane Handbook and presented a descriptive analysis because of the high heterogeneity found across the included studies. We searched the Cochrane Central Register of Controlled Trials, PubMed, and EBSCO Host databases, as well as the grey literature, between 1990 and April 2021. No language restrictions were applied. Only randomised clinical trials (RCTs) were selected. Results: Of the total of 364 interventions included in the eleven RCTs evaluated, 11 (3%) were related to healthcare structure, 51 (14%) to the healthcare delivery process and 302 (83%) to patient outcomes. Regarding the impact of using the EB-CPGs, in 303 interventions (83%), there were no significant differences between the control and experimental groups. In 4 interventions (1%), the result favoured the control and intervention groups in 57 of the interventions (16%). Conclusions: Our study showed that EB-CPGs slightly enhanced the quality of health care in the three dimensions described by Donabedian. Future RCTs should improve their design and methodological rigour by considering the certainty of the evidence supporting the EB-CPGs recommendations. In that context, broader analyses could be performed, having more concise hypotheses for further research. Registration: PROSPERO CRD42020205594",
"keywords": [
"Clinical Practice Guidelines",
"CPG",
"effect",
"health care quality."
],
"content": "Introduction\n\nThe emergence of the Evidence-Based Clinical Practice Guidelines (EB-CPGs) in the 1990s has improved decision-making for healthcare personnel and patients with different health conditions (IOM, 1990; Weisz et al., 2007).\n\nWorldwide, significant efforts have been made to develop and implement EB- CPGs based on scientific evidence. The term “evidence-based” means that the recommendations described in the CPGs derive from the best scientific findings and the highest quality of evidence obtained from applying unbiased, transparent, and rigorous methods to support clinical care (Watters, 2008; Linskey, 2010).\n\nEB-CPGs are statements that include recommendations intended to improve the quality and consistency of health care (Woolf et al., 1999; Kwan, 2004) and to assist clinical staff and patients in the decision-making process (Alonso-Coello et al., 2010; IOM, 2011).\n\nThis study corresponds to the second systematic review of a series of studies on different clinical subjects, which aim to assess the success of the use of EB-CPGs (Ramírez-Morera et al., 2019). Therefore, we evaluated the effectiveness of the application of EB-CPGs for the improvement of the quality of health care in three dimensions: structure, process, and patient outcome in the management of breast cancer (Donabedian Model, Donabedian, 1988).\n\nStudies measuring the effects of EB-CPGs on the quality of health care have mainly focused on the effects on clinical practice (Lugtenberg et al., 2009), state that some international reviews have demonstrated that most of the studies have resulted in significant improvements to the process of care. However, few studies have focused on the effects of measures on patients’ health outcomes.\n\nWe considered for this review breast cancer disease because this is the most common malignancy in women around the world (Ghoncheh et al., 2016). Although survival has improved in the last 30 years mainly because of the implementation of early detection programs, it still registers 2.3 million new diagnostics in women during 2020 and 685 000 deaths within the same year (WHO, 2020).\n\nStrategies to control and prevent this type of cancer must be a high priority for health policymakers (Ghoncheh et al., 2016). According to the above, hundreds of breast cancer guidelines have been published worldwide to reduce its negative impact on men’s and especially on women’s health.\n\nThis review is relevant because there is a growing number of EB-CPGs in different essential areas, and their actual impact on relevant outcomes needs to be assessed. Few systematic reviews evaluate the effect of EBGPC in improving health care; these focus solely on one clinical entity and cover a country or region (Grimshaw et al., 1993; Worrall et al., 1997; Lugtenberg et al., 2009; Ricci-Cabello et al., 2020). For this reason, we reviewed the evidence on the benefits of implementing EB-CPG to improve the quality of care. This review responds to the need for EB-CPG research synthesis on the overall quality of health care delivery.\n\nThis systematic review contributes to meeting the need for research synthesis about EB-CPG by assessing the overall quality of health care delivery. Besides, we visualise the need for a systematic review that conclusively demonstrates the pragmatic impact that evidence-based recommendations have on breast cancer patients.\n\n\nMethods\n\nWe conducted a systematic review to identify and analyse the effect of EB-CPGs on health care quality improvement within the Donabedian Model dimensions: structure, process, and results (Donabedian,1988). We followed the Cochrane Handbook methodological recommendations described by Higgins et al. (2022a).\n\nThis review is registered at PROSPERO (ID: CRD42020205594).\n\nThe research question was translated into the PICO framework for guiding the study search and the criteria selection (Table 1). We developed a method to incorporate the methodological component of the search strategy combined with selected index and free-text terms.\n\nWe explored the following electronic databases for primary studies: Cochrane Central Register of Controlled Trials (CENTRAL), Cochrane Library, including the Cochrane Effective Practice and Organisation of Care (EPOC) group specialised register, Pubmed, Scopus, EBSCO, Academic Search Complete, CINAHL, Biomedical Reference Collection: Comprehensive, APA PsycInfo, Nursing & Allied Health Collection: Comprehensive, Alt HealthWatch, SPORTDiscus with Full Text, Psychology and Behavioral Sciences Collection, Health Source: Nursing/Academic Edition, Biomedical Reference Collection: Basic, AMED - The Allied and Complementary Medicine Database, Consumer Health Complete, Cochrane Database of Systematic Reviews, Cochrane Methodology Register, Rehabilitation & Sports Medicine Source, AgeLine, Global Health, International Pharmaceutical Abstracts, MasterFILE Premier, Rehabilitation & Sports Medicine Source, LILACS, and Health Technology Assessment Database. We also searched the Science Citation Index and Social Sciences Citation Index for papers that refer to studies included in the review.\n\nThe PubMed search strategy was executed in the other databases using the appropriate controlled vocabulary. Searching for other resources included grey literature from different sources and hand searching of those high-yield journals and conference proceedings that have not already been hand searched on behalf of the Cochrane Collaboration.\n\nAuthors of relevant papers were contacted regarding any further published or unpublished work. Authors of other reviews in the field of effective professional practice were contacted regarding relevant studies of which they may be aware. We searched for studies published between January 1990 and April 2021. The search strategy was not restricted by language. An advanced search strategy and results are available as extended data, Appendix 1 (Ramírez-Morera et al., 2022).\n\nFor the management of bibliographic references of the articles found, the web application “Sciwheel Reference Manager & Generator” was used (Sciwheel, 2022).\n\nThe studies found through the search strategy were screened by two reviewers (AR, JS), and discrepancies about study selection were resolved by a third reviewer (MT). Inclusion criteria were: 1. Randomised Clinical Trial (RCT) or cluster-type RCT measuring the impact of using any implementation model versus passive dissemination or no use of the EB-CPG. 2. The studies evaluated the impact on any of the three domains described in the Donabedian model (structure, process, and patient outcomes) for using EB-GPC in treating breast cancer. 3. No language restriction. 4. Published studies from 1990 to 2021.\n\nThree authors (AR, JS, ALR) independently undertook data extraction. They used a modified version of the Cochrane Collaboration EPOC Group “Data Collection Checklist”, employing an electronic datasheet (EPOC, 2019).\n\nTo assess the bias risk, we used standard Cochrane methods described in chapters 8, 10 and 23 of the Cochrane Handbook for Systematic Reviews of Interventions (Higgins et al., 2022b; Sterne et al., 2019). In the case of RCTs, bias resulting from several types of systematic errors was assessed according to methods described by Cochrane (Higgins et al., 2022c) and following the RoB2 instrument (Sterne et al., 2019).\n\nAll studies deemed eligible for the review were assessed independently by the review authors (AR, MT), and discrepancies were resolved by discussion. A summary of the risk of bias assessment is presented as part of the characteristics of included studies table.\n\nWe did not find cluster-type RCTs; then, we did not apply what is stated in chapter 23 of the Cochrane Handbook for Systematic Reviews of Interventions (Higgins et al., 2022d) about assessing cluster RCT following the RoB2 instrument (Sterne et al., 2019).\n\nAn analysis of the quality of the evidence related to each of the outcomes was performed using the GRADE approach (Schünemann et al., 2013). We assessed the certainty of the body of evidence for each key outcome as “high”, “moderate”, “low”, or “very low”, using the GRADEpro GDT platform (GRADEpro, 2021).\n\nData were analysed using Review Manager software, version 5.4 (RevMan, 2020). Revman default templates for data extraction were modified to show the results in a simplified way.\n\nWe found very high variability between the measures of effect within the included studies in this review. Then, we decided not to perform a meta-analysis and, therefore, neither to measure statistical heterogeneity.\n\n\nResults\n\nThe process describing the analysis of studies retrieved through the systematic search is content in the PRISMA flowchart (Figure 1, Page et al., 2021). We retrieved 25002 studies from database searching and 20 studies were found from additional sources identified. We excluded 15900 duplicated records, and 6072 studies were excluded after screening by title and abstract. We assessed 83 articles at the full-text level, excluding 72 references which did not meet the selection criteria: 51 (71%) were not randomised controlled trials, and 20 (28%) did not evaluate clinical practice guidelines.\n\n* The appendix 1 shows the number of records identified from each database or register searched.\n\n** All records were excluded by the authors. Excluded after screening by Title/Abstract.\n\n*** The records were not randomised controlled trials.\n\n**** The records did not evaluate clinical practice guidelines.\n\n***** They published the results included by Irene et al. (2019), whose study described a broader methodology and reported more detailed results.\n\nFrom: Page MJ, McKenzie JE, Bossuyt PM, Boutron I, Hoffmann TC, Mulrow CD, et al. The PRISMA 2020 statement: an updated guideline for reporting systematic reviews. BMJ 2021;372:n71. doi: 10.1136/bmj.n71. For more information, visit: http://www.prisma-statement.org/\n\nStark et al. (2018) was excluded from this systematic review. They published the results included by Irene et al. (2019), whose study described a broader methodology and more detailed reported results. The list of excluded studies and reasons for exclusion are available as extended data, Appendix 2 (Ramírez-Morera et al., 2022).\n\nWe included 11 RCTs analysing CPGs for breast cancer (Boekhout et al., 2015; Greenlee et al., 2016; Grunfeld et al., 2011; Hershman et al., 2013; Irene et al., 2019; Klinkhammer-Schalke et al., 2012; Knobf et al., 2016; Maly et al., 2017; Park et al., 2015; Park et al., 2019; Smith-Turchyn et al., 2020). The list of studies selected after screening and assessing the full text is available as extended data, Appendix 3; Ramírez-Morera et al., 2022).\n\nThe trials were published between 2011 and 2020, most from 2014 to 2016 (5, 46%). Approximately 55% (6) were carried out in the United States of America. The clinical practice guidelines examined within the selected trials were follow-up (11, 100%) and treatment (4, 36%). When evaluating the outcome categories involving the clinical practice guidelines quoted, most referred to the quality of life (7, 63%). A summary of the characteristics of the included studies (n=11) is available (Table 2). A broader description of these characteristics is available as extended data (Appendix 4; Ramírez-Morera et al., 2022) and the list of the CPGs examined in the included studies (Appendix 5; Ramírez-Morera et al., 2022).\n\n* Percentages exceed 100% because the categories are not mutually exclusive (i.e., some studies involved more than one type of guideline scope and more than one outcome category).\n\nTwo different studies (Maly et al., 2017; Irene et al., 2019) evaluated the CPG for Fertility preservation for patients with cancer: American Society of Clinical Oncology clinical practice guideline update (Loren et al., 2013). Park et al. (2015) and Smith-Turchyn et al. (2020) analysed the American College of Sports Medicine roundtable on exercise guidelines for cancer survivors (Schmitz et al., 2010).\n\nAdditionally, Maly et al. (2017) examined the latest version of the CPG by Khatcheressian et al. (2013), American Society of Clinical Oncology 2006 update of the breast cancer follow-up and management guidelines in the adjuvant setting, previously analysed by Hershman et al. (2013) and Greenlee et al. (2016). They assessed Khatcheressian et al. (2006).\n\nGrunfeld et al. (2011) reported the results until 12 months. Extended results (up to 24 months) for the same study were published by Boekhout et al. (2015). Hershman et al. (2013) and Greenlee et al. (2016) published different results of the same study due to the questionaries or scale utilised for measuring outcomes. The first was more interested in the quality of life and treatment satisfaction, and the second in lifestyle behaviours. We found that Knobf et al. (2016) and Park et al. (2019) published the same study. Knobf et al. (2016) focused on describing the effect of exercise on bone density, while Park et al. (2019) described the results concerning the quality of life.\n\nRisk of bias assessment\n\nWe assessed the risk of bias with the RoB2 instrument in the eleven included RCTs following the methods described.\n\nWe found that a low risk of bias prevailed (6; 55%) in domain 1: randomisation process. Some concerns occurred in domain 2: deviations from the intended interventions (7; 64%). A low risk of bias was found for domain 3: missing outcome data (10; 91%) and domain 4: measurement of the outcome (8; 73%). Finally, we found some concerns in domain 5: selecting the reported result (7; 64%).\n\nOverall, one study (9%) reported low risk (Irene et al., 2019), some concerns arise from 6 (55%) studies (Grunfeld et al., 2011; Hershman et al., 2013; Boekhout et al., 2015; Park et al., 2015; Greenlee et al., 2016; Smith-Turchyn et al., 2020) and high risk occurred in 4 (36%) studies (Knobf et al., 2016; Klinkhammer-Schalke et al., 2012; Maly et al., 2017; Park et al., 2019). A summary of the results is available in Figure 2.\n\nD1: Randomisation process. D2: Deviations from the intended interventions. D3: Missing outcome data. D4: Measurement of the outcome. D5: Selection of the reported result. Overall risk of bias.\n\nMost of the risk of bias found in the included studies occurred due to the lack of existence or some level of blinding. In some cases, outcome data were not available entirely for all randomised participants. Four studies did not analyse the intention to treat, and some outcome measurement methods were not adequately described. A broader description of the risk of bias assessment is available as extended data in Appendix 6 (Ramírez-Morera et al., 2022).\n\nThe studies showing the lowest risk of bias (Irene et al., 2019) and one resulting in the highest risk of bias (Park et al., 2019) were chosen to be evaluated with the GRADE methodology and to build a summary of the findings. We decided to grade only four outcomes per study described, including two reporting statistically significant results and two not statistically significant results. Performing a GRADE table provided the rank of possible grades for the certainty of the evidence found in the 362 interventions from the 11 studies included.\n\nThe results varied from high to very low certainty of the evidence, according to the GRADE classification. We found several types of systematic errors in the studies: random sequence generation (selection bias, in 4 studies), incomplete outcome data (attrition bias, in 3 studies), and imprecision (observed within all the studies, in 303 outcomes evaluated representing 83% of the total).\n\nWe found for the study with the lowest risk of bias (Irene et al., 2019) a range for the certainty of the evidence between high (for fertility-related concerns scale scores ≤3 and improvement with no or low fertility or pregnancy concerns) to moderate certainty of the evidence (for improvement in at least one women’s health issue and 50% decrease in the hot flash score), as described in Table 3a.\n\na Risk of bias. Lack of blinding in clinical staff.\n\nb Imprecision. 95% CI: 1.10 to 4.84. p: 0.03. Statistically significant.\n\nc Imprecision. 95% CI: 1.12 to 6.29. p: 0.03. Statistically significant.\n\ne Imprecision. 95% CI: 0.99 to 3.40. p: 0.057. Not statistically significant. Downgraded -1 for imprecision.\n\nf Imprecision. 95% CI: 0.57 to 2.18. p: 0.75. Not statistically significant. Downgraded -1 for imprecision.\n\nh Strong association. OR > 2. Large effect. Upgraded +1.\n\nIn the case of Park et al. (2019) study reporting a high risk of bias, we found a range for the certainty of the evidence between low (moderate MET-min/wk and walk MET-min/wk at six months) and very low certainty of the evidence (Moderate MET-min/wk and Walk MET-min/wk at 12 months), as described in Table 3b. Our results after grading the certainty of the evidence for both studies were consistent with the findings from the RoB2 instrument.\n\na Risk of bias. Lack of allocation concealment. Selection bias. Lack of blinding. Downgraded -2 for risk of bias.\n\nb Imprecision. 95% CI: 606.091 to 690.069. p: 0.0004. Statistically significant.\n\nc Imprecision. 95% CI: 32.289 to 122.711. p: 0.67. Not statistically significant. Downgraded -1 for imprecision.\n\nd Imprecision. 95% CI: 373.268 to 457.592. p: 0.02. Statistically significant.\n\ne Imprecision. 95% CI: 238.718 to 328.562. p: 0.12. Not statistically significant. Downgraded -1 for imprecision.\n\nThe outcomes were grouped in simple relative and absolute numbers. A global estimate of the measurements of the effects included in the studies is lacking because of the significant variability of measuring units combined with the clinical heterogeneity found among the studies included.\n\nThere was significant variability in the measurement of the outcomes reported in the studies. Most were continuous, e.g., to assess the quality of life, which corresponds to the dimension of patient outcome. A total of 362 were included in the 11 RCTs evaluated; 11 (3%) corresponded to the health care structure dimension, 51 (14%) interventions to the dimension process and 302 (83%) interventions to the dimension of patient outcomes. A broader description of the main findings by the dimensions evaluated in the included studies is available as extended data, Appendix 7 (Ramírez-Morera et al., 2022).\n\nRegarding the impact of using EB-CPG, we found 303 (83%) interventions with no significant difference between the control and experimental groups. The outcome favoured the control group in 4 (1%). Three outcomes interfered with the patient’s adherence (predictable variables: age, marital status, and hot flashes), as reported by Maly et al. (2017). Also, the fourth outcome informed by Klinkhammer-Schalke et al. (2012) reported rates of therapeutic options for Physiotherapy 16 (experimental group) vs 30 (control group), p: <0.02, at six months. The result favoured the intervention group for 57 interventions (16%) (Table 4).\n\n\nDiscussion\n\nFor more than two decades, governmental and non-governmental institutions have been making economic and methodological efforts to develop more and better quality EB-CPGs, seeking to deal with different issues most healthcare systems face. Such as the ageing population, rising costs motivated by increased demand for quality care, increasingly expensive emerging health technologies, variability in the provision of health by presuming that part of this disparity could cause inadequate care (either overuse or underuse of supplies), and the desire for clinicians and patients to provide and to receive, respectively, the best possible care with measurable clinical effect. However, it still appears that some of these EB-CPGs are far from contributing to an effective, standardised clinical practice based on the best available evidence (Woolf et al., 1999; IOM, 2011; Alonso-Coello et al., 2010). We agree with Woolf et al. (1999) that EB-CPGs that promote proven benefits and discourage ineffective interventions could reduce morbidity and mortality, and improve quality of life, at least for some conditions. EB-CPGs can also improve the consistency of care.\n\nThe effects of the recommendations in the interventions included in the 11 RCTs considered in this project were in the structure of medical care (3%) and the care provided (14%); both were the least explored. Surprisingly, patient outcomes were the most evaluated domain (83%), with significant results in 43 of 302 (14%), representing 75% of all significant results.\n\nThis fact could lead us to suppose that researchers finally focused on the importance of evaluating the patient health, solely prioritising evaluating the adherence to the CPG (assess the process dimension). They are trying to glimpse more clearly what the use of the CPG represents for patients and not only for clinical staff. As this review considered breast cancer, we could not ignore that there is more social and economic pressure to know the patient outcome.\n\nGrimshaw and Russell (1993) findings described in their systematic review, “Effect of clinical guidelines on medical practice: a systematic review of rigorous evaluations”, reported more than 80% significant improvements among the included studies. Contrasting their results, in this second systematic review, we found 57 interventions on breast cancer in favour of the use of EB-CPGs distributed in all the studies included (16% of all the interventions evaluated). Then, compared to our first systematic review, only half of the measures with statistically significant results favour using EB-CPGs (Ramírez-Morera et al., 2019). However, as 75% of these results were found for the dimension of results in patients, we keep optimistic about the finding on incoming reviews an increasingly more significant impact.\n\nFour studies (36%) reported a high risk of bias (Knobf et al., 2016; Klinkhammer-Schalke et al., 2012; Maly et al., 2017; Park et al., 2019), and some concerns arise from 6 (55%) studies (Grunfeld et al., 2011; Hershman et al., 2013; Boekhout et al., 2015; Park et al., 2015; Greenlee et al., 2016; Smith-Turchyn et al., 2020). Because of that, we agree with Ivers et al. (2012) that EB-CPGs must be evaluated, including more remarkable methodological quality studies to provide feedback and corrective measures for clinical practice through audits, promoting improvements in the quality of care.\n\nWe also concur with Lugtenberg et al. (2009) about the need to focus on the strength of the recommendations for determining what factors influence the use of the guidelines and the improvement of the results of the patients.\n\nThen we identified the need to perform recommendations distinguishing between the level of certainty of evidence (stratified analysis) as this could have a more significant impact on the results when the best available certainty of evidence recommendation is implemented. We keep in mind that explicit EB-CPG improves clinical practice when introduced within a context of rigorous evaluations (Grimshaw & Russell, 1993; Ricci-Cabello et al., 2020).\n\nSome studies included in the review five reported outcomes in the process area (Boekhout et al., 2015; Grunfeld et al., 2011; Irene et al., 2019; Klinkhammer-Schalke et al., 2012; Maly et al., 2017). All of them described favourable results for the intervention in 14 of 51 measurements (27%). This fact reflects that researchers have continued to endeavour to measure when EB-CPGs should be used or not, but this time to a much lesser extent (14% vs 64%) when compared to the first review.\n\nLugtenberg et al. (2009) reported that the size of the effects observed in their systematic review varied considerably between the recommendations within the guidelines. We repeated this finding in our study and the previous one (Ramírez-Morera et al., 2019). We found that in many evaluated interventions (303, 83%), the use of EB-CPG did not reflect any impact in any dimension. The approach followed to report the current effectiveness of EB-CPGs remains incomplete (Woolf et al., 1999), and a strategy that captures better results has not yet been found.\n\nEB-CPGs may contribute to improving the quality of healthcare. However, it is still necessary to integrate them with strategies that enhance their use and effect, such as academic and educational visits as part of ongoing training programs (O’Brien et al., 2007). Repeatedly, advocates for EB-CPGs consider their only existence as a magic solution to solve health care problems; however, they ignore other practical actions that should be implemented along with the guidelines (Woolf et al., 1999).\n\nEB-CPG has an essential role when clinicians do not clearly know the appropriate practice and which scientific evidence should support their decisions (Woolf et al., 1999). Then, EB-CPG developers should be vigilant in identifying these needs to help close this information gap and increase the enthusiasm for employing them.\n\n\nConclusions\n\nDeveloping strategies for a more standardised implementation of the EB-CPGs through structured programs within health systems is essential. Improving the awareness of clinical staff about the possibility of enhancing clinical practice and patient outcomes by using evidence-based recommendations with an expected effect is a need.\n\nThere is an imbalance between the number of EB-CPGs developed for breast cancer and the number of high-quality studies evaluating their effectiveness. Due to the limited results found on the benefit of using EB-CPG, we must continue investigating the subject. We could gradually structure a more robust hypothesis about the variables influencing this issue.\n\nThe variation in the effects found for the recommendations included in the EB-CPGs suggests that it would be helpful to change strategies and focus on the analysis of the limitations of adherence and on designing implementation approaches adapting each recommendation.\n\nIn addition, future RCTs should distinguish the levels of certainty of the evidence supporting each recommendation in their evaluations. Researchers should focus on evaluating recommendations expected to have the most significant impact (superior levels of certainty of the evidence: High or Moderate).\n\nMore research is necessary to define which factors related to the implementation of EB-CPG and its specific recommendations are essential to predict the application of EB-CPG and, therefore, achieve better patient results.\n\nThis systematic review aimed to support the development of programs evaluating the effects of EB-CPG on the quality of health care. Also, to provide reliable evidence sustaining the decision-making process related to the production of EB-CPGs.\n\nEven when some of the results of this systematic review were statistically significant, supporting the use of EB-CPG as a tool to improve clinical practice and quality of care, the results of this review need to be interpreted with caution.\n\nThe implementation of EB-CPGs must consider the differences in the measures of effect to define customised approaches and specific recommendations within the guideline to enhance health care.\n\nFor the adequate implementation of EB-CPGs, it is necessary to consider the possible costs, risks, and benefits and the expected effects derived from EB-CPGs recommendations. The efforts to build EB-CPGs must be complemented with psychosocial strategies promoting health personnel to follow the recommendations of the EB-CPG and evaluate their impact.\n\nGreater methodological rigour in the development of CPGs is needed. It is also required to carry out this process within a standardised formal program in the health systems. Greater credibility could be achieved if recommendations are based on the best available evidence, improving the credibility of their positive effect among health personnel. It could also lead to a more willingness to implement the CPGs and to participate in evaluating their impact.\n\nThis study corresponds to the second systematic review of a series of studies aiming to assess the effect of Evidence-Based Clinical Practice Guidelines (EB-CPGs) on improving health care quality. Our next and last project will investigate Covid-19 disease.\n\nDue to recent research in this field, and the results of this study were not conclusive, more research is necessary to evaluate how EB-CPGs could impact the quality of health care, especially emphasising fewer investigated areas, such as the structure of healthcare services and patient outcomes.\n\nAdditionally, the design and methodological rigour applied to future RCTs should improve by considering the certainty of the evidence supporting the EB-CPGs recommendations. Besides, focusing on those with a greater level of evidence (high or moderate) which could lead to determining more clearly the effect that these recommendations have on the quality of health care.\n\n\nData (and software) availability\n\nUnderlying data\n\nAll data underlying the results are available as part of the article, and no additional source data are required.\n\nExtended data\n\nOpen Science Framework: Extended data for the second SR CPG Breast Cancer. DOI: https://osf.io/6h9pm/?view_only= (Ramírez-Morera et al., 2022).\n\nThis project contains the following extended data:\n\nAppendix 1. Advanced search strategy and results.pdf\n\nAppendix 2. List of excluded studies and reasons for exclusion.pdf\n\nAppendix 3. List of selected studies after screening and assessing the full text.pdf\n\nAppendix 4. Characteristics of the included studies.pdf\n\nAppendix 5. List of the CPGs examined in the included studies.pdf\n\nAppendix 6. Risk of Bias 2 assessment.pdf\n\nAppendix 7. Main findings by dimensions of the included studies.pdf\n\nReporting guidelines\n\nWe followed the PRISMA 2020 statement for reporting systematic reviews (Page et al., 2021). We did the PRISMA 2020 checklist and the flow diagram for new systematic reviews, which included searches of databases, registers, and other sources.\n\nOpen Science Framework: PRISMA checklist and flow chart for Effects of evidence-based clinical practice guidelines for breast cancer in health care quality improvements. A second systematic review. DOI: https://osf.io/k7f5x/?view_only=7d6b4a63853c43e4b233424ea226e66a.\n\n\nAuthor contributions\n\nAnggie Ramírez-Morera: Conceptualisation, Data Curation, Formal Analysis, Funding Acquisition, Methodology, Project Administration, Writing – Original Draft Preparation, Writing – Review & Editing.\n\nMario Tristán: Conceptualisation, Data Curation, Formal Analysis, Funding Acquisition, Methodology, Resources, Software, Supervision, Writing – Original Draft Preparation, Writing – Review & Editing.\n\nJordan Salazar-Vargas: Data Curation, Formal Analysis, Funding Acquisition, Resources, Writing – Original Draft Preparation, Writing – Review & Editing.\n\nAna Leonor Rivera-Chavarría: Data Curation, Writing – Original Draft Preparation, Writing – Review & Editing.\n\n\nLeading author information\n\nAnggie Ramírez-Morera is PhD candidate Program in Biomedical Research Methodology and Public Health, Universitat Autònoma de Barcelona.",
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PubMed Abstract | Publisher Full Text\n\nStark SS, et al.: Randomised controlled trial of the effect of a reproductive health survivorship care plan on fertility and pregnancy concerns, vasomotor symptoms, sexual health, and contraception in young breast cancer survivors. Fertil. Steril. 2018; 110(4): e48. Publisher Full Text\n\nWatters W: Defining evidence-based clinical practice guidelines. “AOOS Now”.2008. American Academy of Orthopaedic Surgeons. RESEARCH2.Reference SourceReference Source\n\nWeisz G, et al.: The Emergence of Clinical Practice Guidelines. Milbank Q. 2007; 85(4): 691–727. PubMed Abstract | Publisher Full Text\n\nWHO: Breast Cancer. Cancer prevention. Early Diagnosis and Screening. Geneva:World Health Organization;2020.Reference Source\n\nWoolf SH, et al.: Clinical guidelines: potential benefits, limitations, and harms of clinical guidelines. BMJ (Clinical Research Ed.). 1999; 318(7182): 527–530. PubMed Abstract | Publisher Full Text | Free Full Text\n\nWorrall G, Chaulk P, Freake D: The effects of clinical practice guidelines on patient outcomes in primary care: a systematic review. Can. Med. Assoc. J. 1997; 156(12): 1705–1712. PubMed Abstract"
}
|
[
{
"id": "154242",
"date": "04 Nov 2022",
"name": "Ignacio Marín León",
"expertise": [
"Reviewer Expertise Clinical Epidemiology"
],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis is an original paper of substantial importance addressing CPG implementation effect. Mostly a very sensible, necessary and well-developed document. In general, the writing is clear and concise, however, needs some editing to avoid unnecessary repetition (i.e. the three domains of Donabedian proposal, and some others). The effect of the implementation of 11 guidelines identified after a rigorous and well-done literature search is presented, as detailed in Methods and Figure 1.\nThe reviewer has some concerns and suggestions regarding Methods (GRADE use) and Discussion that need to be addressed to improve the manuscript.\nAn article that is worthy of being indexed.\nMajor Comments\nMethods:\nData extraction, page 4, last paragraph, “An analysis of the quality of the evidence…we assessed the certainty...”: It is unclear how this was done. GRADE is a very labour-intensive and specific methodology, and to perform a GRADE evaluation, the authors have two options: a) simply seek the strength of evidence from the original implemented guideline included in the review, or b) evaluate and critically assess original articles cited in the guidelines reviewed, when they did not use GRADE methodology. This needs clarification on what the authors mean. In addition, readers do not need the authors to explain what GRADE method is.\nResults: Quality of evidence assessment, page 7: This section should be clarified in accordance with previous comments on the use of GRADE.\nDiscussion:\nThe structure of this section does not agree with the established \"norms\" on how the \"discussion\" section should be drafted. The main findings of the study should first be highlighted in relation to the objective of the study, and in a second step explain the causes of such findings and contrast with similar studies in the available literature. Thus, the large first paragraph could be moved down (and shortened) in the text.\n\nThe reviewer does not agree with the statements made in the current third paragraph, in the sense of contrasting the effect on the \"process of care\" with the effect on \"patient outcomes\". There are RCTs that relate the implementation of guidelines process of care with the appropriateness and necessity of care for patients' benefits (i.e. see Romero et al. (2005)1).\n\nSome comment on study weaknesses is missing.\n\nMinor Comments:\nAbstract: It is required that the clinical problem addressed in the study (breast cancer) appears in the abstract and keywords.\nIntroduction: In the sixth paragraph: “Although survival has improved…..”: There is a huge debate whether the improved survival depends on the early diagnosis or the improvement of knowledge of the natural history and new treatments of breast cancer (Gøtzsche et al. (2013)2, Elmore et al. (2005)3). Thus, some sentences should be complemented with “...of early detection programs and treatment improvement”.\nDiscussion:\nThe comments on the usefulness of EB-CPG could be shortened.\n\nAre the rationale for, and objectives of, the Systematic Review clearly stated? Yes\n\nAre sufficient details of the methods and analysis provided to allow replication by others? Partly\n\nIs the statistical analysis and its interpretation appropriate? Yes\n\nAre the conclusions drawn adequately supported by the results presented in the review? Yes",
"responses": [
{
"c_id": "9083",
"date": "19 Dec 2022",
"name": "Anggie Ramirez",
"role": "Author Response",
"response": "Dear Dr. Ignacio Marin, We would like to thank you for agreeing to participate in the review of our research. We consider that your contributions are very valuable to improve our work. Regarding your comments, we describe as follows the research team's position on each of them: A. Concerning the analysis of the certainty of the evidence, you wrote: Methods: Data extraction, page 4, last paragraph, \"An analysis of the quality of the evidence…we assessed the certainty...\": It is unclear how this was done. GRADE is a very labour-intensive and specific methodology, and to perform a GRADE evaluation, the authors have two options: a) simply seek the strength of evidence from the original implemented guideline included in the review, or b) evaluate and critically assess original articles cited in the guidelines reviewed, when they did not use GRADE methodology. This needs clarification on what the authors mean. In addition, readers do not need the authors to explain what GRADE method is. Results: Quality of evidence assessment, page 7: This section should be clarified in accordance with previous comments on the use of GRADE. Answer to #A: We agree with you about the options using the GRADE method to develop or adapt a Clinical Practice Guideline. However, we used the GRADE method to assess the certainty of the evidence only for the RCTs that met the inclusion criteria. Therefore, it does not comprise part of the objective and scope of this systematic review to evaluate the evidence included in the CPGs that these studies reviewed. We wrote as part of the Methods section: The studies showing the lowest risk of bias (Irene et al., 2019) and one resulting in the highest risk of bias (Park et al., 2019) were chosen to be evaluated with the GRADE methodology and to build a summary of the findings. We decided to grade only four outcomes per study described, including two reporting statistically significant results and two not statistically significant results. Performing a GRADE table provided the rank of possible grades for the certainty of the evidence found in the 362 interventions from the 11 studies included. Hence, we do not consider it necessary to modify the text in the section \"Quality of evidence assessment\". Then, we want to clarify that it is part of the objective and scope of this systematic review to evaluate the certainty of the evidence of the RCTs included and not of the EB-CPGs that they evaluated. B. About the article's Discussion section, you wrote: B.1 The structure of this section does not agree with the established \"norms\" on how the \"discussion\" section should be drafted. The main findings of the study should first be highlighted in relation to the objective of the study, and in a second step explain the causes of such findings and contrast with similar studies in the available literature. Thus, the large first paragraph could be moved down (and shortened) in the text. Answer to #B.1: It is relevant to clarify that our article complies with F1000Research guidelines for preparing a Systematic Review article. The journal requests the main body section as follows: Main Body: The format of the main body of the article is flexible: it should be concise, making it easy to read and review, and presented in a format that is appropriate for the type of study presented. For most Systematic Reviews, the following standard format will be the most appropriate: Introduction, Methods, Results, Conclusions/Discussion. Also, we consider that the aim of the discussion was achieved because we highlighted our findings and contrasted them with similar research. We must add that we have also complied with the PRISMA 2020 statement checklist (Page et al., 2021[1]). About the discussion, our study meets with the following items: 23a Provide a general interpretation of the results in the context of other evidence. 23b Discuss any limitations of the evidence included in the review. 23c Discuss any limitations of the review processes used. 23d Discuss the implications of the results for practice, policy, and future research. Therefore, considering that neither of the indications describes an expected order of ideas as you have indicated, the authors alleged it convenient to include this first paragraph to provide a context about the world situation regarding the efforts to develop guidelines and the expectations placed by health caregivers on their effect. Also, to link this finding with the findings in our study. Consequently, as the first paragraph of this section is congruent with the aim of this systematic review, we consider it appropriate to keep the paragraph unchanged. B.2 Additionally, regarding your comment about the third paragraph of the discussion section: The reviewer does not agree with the statements made in the current third paragraph, in the sense of contrasting the effect on the \"process of care\" with the effect on \"patient outcomes\". There are RCTs that relate the implementation of guidelines process of care with the appropriateness and necessity of care for patients' benefits (i.e. see Romero et al. (2005)[2]). We would like to explain the following: Answer #B.2: This statement refers to the findings we got from the first systematic review (Ramírez-Morera et al., 2019[3]) that focused on the impact of CPGs in the cardiovascular approach; at that time, we included nine RCTs. Therefore, we decided to compare the results obtained in this second SR to our previous research regarding the number of interventions evaluated according to the dimensions described by Donabedian (1988)[4]. This report corresponds to the result of a Systematic Review that includes only Randomized Controlled Clinical Trials (RCTs) whose objective was to measure the impact that Clinical Practice Guidelines have produced on the quality of health care. This review's authors analyzed the RCT measurements' results according to the framework proposed by Avedis Donabedian[5] (7 January 1919 – 9 November 2000). Therefore, what has been understood by the implementation of clinical practice guidelines, as defined in the UK glossary published by NICE, adheres to the definition mentioned earlier: \"implementation, The process of putting guideline recommendations into practice\" (NICE 2014)[6]. Evaluation components of CPG use, such as clinical auditing, can be of great value. However, clinical auditing has not been disseminated to be available in other countries, predominantly low-income countries. In 2008, Dutch authors Lugtenberg M, Burgers JS and Westert GP published an excellent systematic review[7] using the quality of care model by Donabedian. This publication has been very inspiring for the authors of this series of systematic reviews. This review mainly includes RCT studies and some before and after studies done on CPG made in the Netherlands, a country with a long tradition in the design and use of CPG. Some of their results are encouraging, although other aspects were weak, such as the impact on patient's health, which should be considered a very relevant result. Due to the nature of the data, it was impossible to have a quantitative synthesis, only qualitative. But it did not inhibit us from assessing the certainty of the evidence for the results of the included studies after searching and screening according to the inclusion criteria. To determine the confidence of the evidence, we have followed Cochrane's recommendations using the tools developed by GRADE. GRADE is a tool that requires knowledge of the essential elements of effective measurement and its potential biases. The authors of this SR are trained to complete this task with adequate competency. B.3 You also wrote: Some comment on study weaknesses is missing. Answer #B.3: We did not write within a specific section of our article what the research's weaknesses are. The weaknesses of the study are described throughout different statements, for example, when describing the risk of bias assessment for the included studies. We also identified many EB-CPGs developed in contrast to the few numbers evaluated through formal RCT-type investigations. We also wrote about the implications for research: Due to recent research in this field, and the results of this study were not conclusive, more research is necessary to evaluate how EB-CPGs could impact the quality of health care, especially emphasising fewer investigated areas, such as the structure of healthcare services and patient outcomes. Additionally, the design and methodological rigour applied to future RCTs should improve by considering the certainty of the evidence supporting the EB-CPGs recommendations. Besides, focusing on those with a greater level of evidence (high or moderate) which could lead to determining more clearly the effect that these recommendations have on the quality of health care. C. About your minor comments: C.1 First, about the abstract section you wrote: Abstract: It is required that the clinical problem addressed in the study (breast cancer) appears in the abstract and keywords. Answer #C.1: We agree to add \"breast cancer\" as a keyword. C.2 Second, concerning the introduction section, you wrote: Introduction: In the sixth paragraph: \"Although survival has improved…..\": There is a huge debate whether the improved survival depends on the early diagnosis or the improvement of knowledge of the natural history and new treatments of breast cancer (Gøtzsche et al. (2013)[8], Elmore et al. (2005)[9]). Thus, some sentences should be complemented with \"...of early detection programs and treatment improvement\". Answer #C.2: We agree to add the suggested text. C.3 Third, referring to the discussion section, you wrote: The comments on the usefulness of EB-CPG could be shortened. Answer #C.3: The authors alleged it is convenient to describe the usefulness of EB-CPG because it is congruent with the aim of this systematic review. Therefore, we disagree with the modification suggested. C.4 Fourth and last, you wrote that there are 'partly' details of the methods and analysis provided to allow replication by others. Answer #C.4: Full details on methods and further analyses to allow replication by others can also be accessed through the research protocol, registered at PROSPERO: CRD42020205594. References [1] Page et al. (2021). The PRISMA 2020 statement: an updated guideline for reporting systematic reviews. BMJ (Clinical Research Ed.), 372, p.n71. [Online]. Available at: doi:10.1136/bmj.n71. [2] Romero A, Alonso C, Marín I, Grimshaw J, et al.: Effectiveness of a Multifactorial Strategy for Implementing Clinical Guidelines on Unstable Angina: Cluster Randomized Trial. Revista Española de Cardiología (English Edition). 2005; 58 (6): 640-648 [3] Ramírez-Morera, A., Tristan, M. and Vazquez, J. C. (2019). Effects of evidence-based clinical practice guidelines in cardiovascular health care quality improvements: A systematic review [version 3; peer review: 2 approved]. F1000Research 2019, 8:1041[Online]. Available at: https://doi.org/10.12688/f1000research.18865.3 [4] Donabedian, A. (1988). The quality of care. How can it be assessed? The Journal of the American Medical Association, 260 (12), pp.1743–1748. [Online]. Available at: doi:10.1001/jama.260.12.1743. [5] Donabedian model. (2022, August 8). In Wikipedia. https://en.wikipedia.org/wiki/Donabedian_model [6] Glossary | Developing NICE guidelines: the manual | Guidance | NICE [WWW Document], 2014 LAST UPDATED 18 January 2022. URL https://www.nice.org.uk/process/pmg20/chapter/glossary [7] Lugtenberg, M., Burgers, J.S., Westert, G.P., 2009. Effects of evidence-based clinical practice guidelines on quality of care: a systematic review. Qual. Saf. Health Care 18, 385–392. doi:10.1136/qshc.2008.028043 [8] Gøtzsche PC, Jørgensen KJ: Screening for breast cancer with mammography. Cochrane Database Syst Rev. 2013. CD001877 [9] Elmore JG, Armstrong K, Lehman CD, Fletcher SW: Screening for breast cancer. JAMA. 2005; 293 (10): 1245-56"
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https://f1000research.com/articles/11-479/v1
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29 Apr 22
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{
"type": "Systematic Review",
"title": "Monitoring of awake bruxism by intelligent app",
"authors": [
"Byron Velásquez Ron",
"Verónica Mosquera Cisneros",
"Pamela Pazmiño Troncoso",
"María Rodríguez Tates",
"Eddy Alvares Lalvay",
"Luis Chauca Bajaña",
"Andrea Ordoñez Balladares",
"Verónica Mosquera Cisneros",
"Pamela Pazmiño Troncoso",
"María Rodríguez Tates",
"Eddy Alvares Lalvay",
"Luis Chauca Bajaña",
"Andrea Ordoñez Balladares"
],
"abstract": "Background. Bruxism is a topic of much controversy and is continually debated in the field of dentistry due to the multifaceted clinical relationship that results in painful conditions and consequences to patients. The aim of this review was to determine the effectiveness of a smartphones app in monitoring awake bruxism. Methods. PROSPERO (registration number: CRD42021271190). The eligibility criteria were as followed: observational studies, case–control\n\nstudies, studies that reported odds ratios, and studies on awake bruxism. The following keywords were searched: [smartphones apps] AND [apps] AND [awake bruxism], OR [sleep bruxism], OR [sleep hygiene], OR [parasomnias], AND [habits]. Results. All the authors agree that the use of the smartphone app allows controlled awake bruxism monitoring. The results also show that\n\nthe two bruxism are interactive, having negative synergism and substantially increasing the risks of temporomandibular joint pain and temporomandibular disorders. Discussion. In the AB it was possible to identify 70% symptoms through the different frequencies of behavior provided by the App, within the present technological tools have become daily in young and adult population. The app is effective and easy to use by patients, effectively limiting biases the time of evaluation.",
"keywords": [
"Awake bruxism",
"self-report",
"ecological momentary assessment",
"smartphone app."
],
"content": "Introduction\n\nThe controversy when talking about bruxism will always be latent among the academy, from a concept of parafunction to a concept of phenomena wherein biological, psychological and exogenous factors act in greater or lesser percentages.1 The independent definitions of day bruxism and night bruxism were pointed out at a meeting of different specialties, with oral rehabilitation experts, maxillofacial surgeons and psychologists, who, in 2020, proposed adequate differentiation between the two.2 Bruxism is a repetitive jaw muscle activity characterized by clenching or grinding of the teeth and/or by bracing or thrusting of the mandible.3,4 “Bruxism has two distinct circadian manifestations: it can occur during sleep (indicated as sleep bruxism) or during wakefulness (indicated as awake bruxism).5 Awake bruxism is currently defined as “masticatory muscle activity during wakefulness that is characterized by repetitive or sustained dental contact and/or reinforcements or pushes of the jaw and is not a movement disorder in healthy individuals”.6\n\nPolysomnography (PSG) and electromyography (EMG) have been used for the evaluation of nocturnal bruxism7; however for the evaluation of awake bruxism (AB), there was no specific evaluator, until 2018 when an app (Manfredini, Bracci, 2018) was created to evaluate and monitor it through the use of smart devices (smartphones).8 Bruxism is not necessarily considered a pathological behavior, but it has clinical consequences, the frequency of AB in the healthy young population allows us to compare with other groups9; in these, psychological factors are determined, including fatigue, muscle pain, tooth wear; having differences between young people and adults differentiating habits and lifestyles that modify the behavior of bruxism.10\n\nThe use of questionnaires (self-reports), such as clinical observation complemented with electromyography (EMG) have helped in the evaluation of awake bruxism; however, the momentary ecological assessment (EMA) combines real-time approaches to the current state of the patient, which facilitates having an objective assessment.11 The limitations of non-instrumental methods to assess the AB are high and become subjective, the use of EMA allows to collect data in real time for a certain period of time according to the coding of alerts, which are activated according to the daily life of the individual,12 the usefulness in the research field is highlighted when evaluating the oral activity of the individual, unfortunately the data obtained are partial, with little research.13,14\n\nTo limit the bias provided by evaluations of the AB, a group of researchers has introduced an app (BruxApp) for smartphones, its foundation of creation is the implementation of EMA, this collects data through alerts (20 daily) with questions of related conditions simple to accept or deny by the individual: teeth in contact, habits, mandibular hypermobility, clenching and grinding of teeth; characteristic signs of AB.15 It is taken as a starting point young population (young adults)16 whom the researchers determine as the control group, it is monitored by the app for a week (20 daily alerts), the frequency was 28.3% in young people with AB with a low coefficient of variation in jaw muscle activity.17 The objective of the present study was to determine the effectiveness of the smartphone app in monitoring awake bruxism. The PICO question was: is the application of smart apps effective in diagnosing daytime bruxism? P: Smartphone patients with the smart app. I: Intervention of all patients with bruxism C: Comparison of bruxism control with the app versus a control group. O: Observation of the percentage of bruxism control.\n\n\nMethods\n\nThis systematic review was registered with PROSPERO under registration number CRD42021271190. The eligibility criteria were as follows: observational studies, case-control studies, studies that reported odds ratios, and studies on awake bruxism. The following keywords were searched using the Boolean operators AND, OR and NOT: [smartphones apps] AND [apps], [awake bruxism], OR [sleep bruxism], OR [sleep hygiene], OR [parasomnias], OR [habits], OR [chewing], OR [teeth grinding], OR [squeezing teeth], OR [parafunctional habits], OR [parafunctional habit], OR [oral habits] OR [oral habit] OR [oral parafunctional] OR [oral parafunctional] OR [oral parafunctional habit] OR [oral parafunctional] OR [oral parafunctional habit] OR [oral parafunctional] OR [parafunctional oral habit] and [Facial pain] OR [temporomandibular joint disorders] OR [Temporomandibular Joint Dysfunction Syndrome] OR [myofascial pain] OR [syndromes] OR [myalgia]] OR [osteoarthritis] OR [pandemic Cov-19] OR [orofacial pain] OR [orofacial pain] OR [TMD] OR [stress] OR [temporomandibular disorder] OR [myofascial pain] OR [disk displacement] OR [young university] OR [young] OR [adult]. The Scopus, EBSCO, PubMed, Medline Embase, Cochrane Library, and Web of Science databases were searched; alternate databases that were searched included Scielo, Latindex, and Redalyc. Using the PRISMA research protocol, the authors used a flowchart to sequentially explain the selected information. The following complete articles published between January 2014 and June 2021 were included: a total of 857 records were obtained; 27 other records were obtained from other sources; 427 duplicate records were deleted; 200 studies were screened; and 102 records were excluded. In total, 98 studies were included in the qualitative analysis, and 16 studies were includedin the quantitative analysis (Figure 1).\n\nThe authors (BVVR, VMC, PP, LCHB, EDL) independently reviewed the titles and summaries, excluded duplicates and irrelevant articles, and considered only full-text articles. The dates and names of all authors in the final review article were included. Any conflict with respect to the inclusion and exclusion criteria was resolved by the third and fourth authors (MRT, AOB). To control for bias, the Scala JADAD (Table 1) was used. The data extraction procedure was evaluated according to the criteria of all authors. Articles were classified by the author/year, study objective, study type, methodology, results (standard mean and deviation) and conclusions.\n\n\nResults\n\nSB is related to nonfunctional occlusion, while AB is related to occlusal interactions, suggesting the need for a different therapeutic approach (Table 2).\n\n\nDiscussion\n\nAll the authors agree that the use of the smartphone app allows controlled AB monitoring by the patient. The current study also showed that the two bruxism are interactive, with negative synergism substantially increasing the risks of TMJ pain and TMD. Signs such as contact between the teeth, clenching of teeth, teeth grinding, and jaw clenching are well defined in the applicationIn AB, it was possible to identify 70% symptoms through the different frequencies of behavior provided by the app, within the present technological tools have become daily in young and adult population.18 In the studies reviewed, the EMA was clear for the entire assigned sample.19,20 In the studies that entered the analysis, the six conditions indicated by the application menu were investigated, relaxed jaw muscles (non-contact teeth), teeth in contact (sander in fixed position), mandibular clenching (without contact between the teeth), dental clenching (strong contact in fixed position), dental grinding and area of pain (temporary, interciliary, temple, preauricular, auricular, mandibular angle, mentonian, neck, frontal, infra and supraorbital),21,22 the data that were obtained were handled by the application menu that allowed to precisely extract a Microsoft Excel file (20 alerts × 7 days) in real time.23 The limitation that was found in the present systematic review is the difficulty of comparing with other studies by the different experimental designs (retrospective), while to apply self-reports are unique times.24 By assessing population behavior frequency is the baseline for observational EMA studies that aids massive data collection,25 it also helps to compare findings related to dietary habits, smoking, medications, psychological pathologies, and comorbid conditions.26 Some studies take as a control group young population Bracci et al. 2018 analyzed healthy young population finding dental contact (13.6%), teeth grinding (0.5%) and relaxed jaw muscles (76.4%), with a combined frequency of AB of (23.6%).26 Some studies take as a control group young population Bracci et al. 2018 analyzed healthy young population finding dental contact (13.6%), teeth grinding (0.5%) and relaxed jaw muscles (76.4%), with a combined frequency of AB of 23.6%.26 These results could be considered a reference point for future research on the epidemiological characteristics of AB in healthy young adults, young people with pathologies, adults and geriatric patients.16,27 The importance of psychological factors was determined, well-defined changes after the COVID-19 pandemic, having been analyzed in AB, the findings were that females are more likely to experience stress, compared with males, the explanation women report better about their emotions28 but the depressive state leads to generate AB crisis with BS in the two genders due to the socio-economic conditions generated by the pandemic, it should be clarified that previous systematic reviews found no gender differences in the frequency of AB29 which contrasts with current information.30 No significant differences were found in the university population, young adults, some authors point out that the monitoring could have been carried out in transition for the student population so that high stress was not indicated, it would be important to develop future research in times such as semester evaluations to determine significant differences.31 It should be considered that the elaboration of the self-report must be controlled, so that unnecessary biases are avoided, for this reason the calibration of the instrument is essential whether individual or group, avoiding or reducing homogeneity to a minimum,32,33 through training and socialization that allows the population to understand the reliable use of self-report based on EMA.34 The characteristics of the populations studied directly influence the results, the age factor, educational level, work activity, socioeconomic status are aspects that influence in substance.34,35 Zanni et al. 2019 found that in one week the relaxation of the mandibular muscles was very low, they conclude that not only in healthy young population the symptoms change from one day to the next,36 the population comportment must be specific, this makes variable the behavior of the AB monitored with the app, recognizing natural fluctuation and difficulty in recognizing the symptoms.37,38 Muscle relaxation can be recognized by the individual, also clenching of teeth,39,40 can be a good reference to evaluate the behavior of AB to be a conscious and controlled activity,41 other authors indicate that the use of SMEs provides reliability in the monitoring of AB, the reason lowers the influence of natural fluctuation that the population presents regardless of age or gender.42 It is recommended to conduct future research that considers long-term monitoring of AB, the hypothesis should be tested that the manifestations of AB: relaxed jaw muscles (non-contact teeth), teeth in contact (sander in fixed position), mandibular clenching (no contact between teeth), dental clenching (strong contact in fixed position), dental grinding and area of pain (temporary, interciliary, temple, preauricular, auricular, mandibular angle, mentonian, neck, frontal, infra and supraorbital, clinical consequences such as temporo mandibular joint dysfunction, regional myalgias43,44 are determined. Continuing with the technological line, the effectiveness of an email-based registration and recovery system should be studied if the individual detects non-functional diurnal contact or muscle contracture, an effective strategy for the treatment of temporo mandibular disorders.45 An assessment of the associated factors and conditions can, in theory, increase or decrease the frequencies of AB behaviors in the app monitored population based on the EMA self-report (e.g., dietary, or smoking habits, medication use, psychological problems, and comorbid conditions).47,48 Data can be added to ongoing studies that consider the 2018 definition of bruxism49 and the refinement of assessment strategies. Comparisons between populations are necessary and can be used in the context of an ongoing multicenter project on the epidemiology of bruxism.50\n\n\nConclusions\n\nThe app used to monitor awake bruxism is effective, and its ease of use allows a fundamental approach to diagnosis.\n\n\nAuthor contributions\n\nVelasquez B: Conceptualization, Data Curation, Formal Analysis, Investigation, Methodology, Project Administration, Resources, Validation, Visualization, Writing Original Draft Preparation. Writing -review & Edith.\n\nAlvarez E.: Conceptualization, Data Curation, Formal Analysis, Investigation, Methodology, Project Administration, Resources\n\nMosquera V: conceptualization, Data Curation, Formal Analysis, Investigation, Methodology, Project Administration, Resources\n\nPazmiño P: conceptualization, Data Curation, Formal Analysis, Investigation, Methodology, Project Administration, Resources\n\nRodriguez M: Conceptualization, Data Curation, Validation, Visualization, Writing – Original Draft Preparation, Writing – Review & Editing;\n\nChauca L: Formal Analysis, Resources, Supervision, Validation, Visualization, Writing – Original Draft Preparation, Writing – Review & Editing\n\nOrdoñez A: Formal Analysis, Resources, Supervision, Validation, Visualization, Writing – Original Draft Preparation, Writing – Review & Editing\n\n\nData availability\n\nNo data are associated with this article.",
"appendix": "Acknowledgments\n\nThe authors would like to express their special thanks to the UDLA (Universidad de Las Americas).\n\n\nReferences\n\nCâmara-Souza MB, Carvalho AG, Figueredo OMC, et al.: Awake bruxism frequency and psychosocial factors in college preparatory students. Cranio. 2020; 14: 1–7. PubMed Abstract | Publisher Full Text\n\nSierwald I, John MT, Schierz O, et al.: Association of temporomandibular disorder pain with awake and sleep bruxism in adults. Journal of orofacial orthopedics = Fortschritte der Kieferorthopadie : Organ/official J Deut Gesell Kieferorthop. 2015; 76(4): 305–317. PubMed Abstract | Publisher Full Text\n\nManfredini D, Arreghini A, Lombardo L, et al.: Assessment of Anxiety and Coping Features in Bruxers: A Portable Electromyographic and Electrocardiographic Study. J. Oral Facial Pain Headache. 2016; 30(3): 249–254. Publisher Full Text\n\nKumar A, Spivakovsky S: Bruxism-is botulinum toxin an effective treatment?. Evid. Bas. Denti. 2018; 19(2): 59. 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PubMed Abstract | Publisher Full Text\n\nManfredini D, Ahlberg J, Aarab G, et al.: Towards a Standardized Tool for the Assessment of Bruxism (STAB)—Overview and general remarks of a multidimensional bruxism evaluation system. J. Oral Rehabil. 2020; 47(5): 549–556. PubMed Abstract | Publisher Full Text\n\nImbriglio TV, Moayedi M, Freeman BV, et al.: Music Modulates Awake Bruxism in Chronic Painful Temporomandibular Disorders. Headache. 2020; 60(10): 2389–2405. PubMed Abstract | Publisher Full Text\n\nElla B, Guillaud E, Langbour N, et al.: Prevalence of Bruxism in Hemifacial-Spasm Patients. J. Prosthodont. 2017; 26(4): 280–283. PubMed Abstract | Publisher Full Text\n\nSierwald I, John M, Schierz O, et al.: Association of temporomandibular disorder pain with awake and sleep bruxism in adults. J. Orofac. Orthop. 2015; 76(4): 305–317. PubMed Abstract | Publisher Full Text\n\nMuzalev K, van Selms M , Lobbezoo F: No Dose-Response Association Between Self-Reported Bruxism and Pain-Related Temporomandibular Disorders: A Retrospective Study. J. Oral Facial Pain Headache. 2018 Fall; 32(4): 375–380. PubMed Abstract | Publisher Full Text\n\nRaphael KG, Santiago V, Lobbezoo F: Is bruxism a disorder or a behaviour? Rethinking the international consensus on defining and grading of bruxism. J. Oral Rehabil. 2016; 43(10): 791–798. PubMed Abstract | Publisher Full Text\n\nMuzalev K, Visscher CM, Koutris M, et al.: Long-term variability of sleep bruxism and psychological stress in patients with jaw-muscle pain: Report of two longitudinal clinical cases. J. Oral Rehabil. 2018; 45(2): 104–109. PubMed Abstract | Publisher Full Text\n\nManfredini D, Ahlberg J, Winocur E, et al.: Management of sleep bruxism in adults: A qualitative systematic literature review. J. Oral Rehabil. 2015; 42(11): 862–874. PubMed Abstract | Publisher Full Text\n\nDe Oliveira M, Almeida A, Felix S, et al.: Sleep bruxism: the complexity of a definitive diagnosis -case report. Ann. Med. 2021; 53(58): S59. Publisher Full Text\n\nCamara M, Guimaraes A, Costa O, et al.: Awake bruxism frequency and psychosocial factors in college preparatory students. Cranio. 2020; 14: 1–7. PubMed Abstract | Publisher Full Text\n\nWetselaar P, Vermaire EJH, Lobbezoo F, et al.: The prevalence of awake bruxism and sleep bruxism in the Dutch adolescent population. J. Oral Rehabil. 2021; 48(2): 143–149. PubMed Abstract | Publisher Full Text\n\nWetselaar P, Vermaire EJH, Lobbezoo F, et al.: The prevalence of awake bruxism and sleep bruxism in the Dutch adult population. J. Oral Rehabil. 2019; 46: 617–623. PubMed Abstract | Publisher Full Text\n\nSomay E, Tekkarismaz N: Evaluation of sleep bruxism and temporomandibular disorders in patients undergoing hemodialysis. Niger. J. Clin. Pract. 2020; 23(10): 1375–1380. PubMed Abstract | Publisher Full Text\n\nZani A, Lobbezoo F, Bracci A, et al.: Smartphone-based evaluation of awake bruxism behaviours in a sample of healthy young adults: findings from two University centres. J. Oral Rehabil. 2021; 48(9): 989–995. PubMed Abstract | Publisher Full Text\n\nReissmann D, John M, Aigner A, et al.: Interaction Between Awake and Sleep Bruxism Is Associated with Increased Presence of Painful Temporomandibular Disorder. J. Oral Facial Pain Headache. 2017; 31(4): 299–305. PubMed Abstract | Publisher Full Text\n\nRofaeel M, Chow JCF, Cioffi I: The intensity of awake bruxism episodes is increased in individuals with high trait anxiety. Clin. Oral Investig. 2021; 25(5): 3197–3206. PubMed Abstract | Publisher Full Text\n\nSerra-Negra JM, Lobbezoo F, Correa-Faria P, et al.: Relationship of self-reported sleep bruxism and awake bruxism with chronotype profiles in Italian dental students. Cranio. 2018; 37(3): 147–152. PubMed Abstract | Publisher Full Text\n\nWinocur E, Messer T, Eli I, et al.: Awake and sleep bruxism among Israeli adolescents. Front. Neurol. 2019; 10(10): 443. PubMed Abstract | Publisher Full Text\n\nLobbezo F, Ahlberg J, Raphael K, et al.: International consensus on the assessment of bruxism: Report of a work in progress. J. Oral Rehabil. 2018; 45(11): 837–844. PubMed Abstract | Publisher Full Text\n\nOsiewicz M, Lobbezoo F, Bracci A, et al.: Ecological Momentary Assessment and Intervention Principles for the Study of Awake Bruxism Behaviors, Part 2: Development of a Smartphone Application for a Multicenter Investigation and Chronological Translation for the Polish Version. Front. Neurol. 2019; 10(10): 170. eCollection 2019. PubMed Abstract | Publisher Full Text"
}
|
[
{
"id": "145993",
"date": "30 Sep 2022",
"name": "Dobromira Antonova Shopova",
"expertise": [
"Reviewer Expertise I am a specialist in prosthetic dentistry and in recent years I have been dealing with modern methods for the treatment of bruxism."
],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe introduction is short, with only 17 cited authors. In general, the number of references is not large - 50, which tends to the minimum for a review article. I ask the authors to add more articles, especially those published in the last 5 years. If desired, they can use some of those that I consider appropriate:\nShopova D, Bozhkova T, Yordanova S and Yordanova M. Case Report: Digital analysis of occlusion with T-Scan Novus in occlusal splint treatment for a patient with bruxism [version 2; peer review: 2 approved]. F1000Research 2022, 10:9151 – It presents a digital device of measurement of occlusal forces, which is very useful in nowadays practice.\n\nShopova D and Mladenov K. Case Report: A digital workflow in the treatment of bruxism in a young patient [version 2; peer review: 2 approved]. F1000Research 2022, 10:8942 – It presents the complete procedure of diagnosis and treatment in case of bruxism only by digital workflow.\n\nBozhkova, T., & Shopova, D. (2021). T-Scan Novus System in the Management of Splints—Pilot Study. European Journal of Dentistry3. – Again, digital method of measurement of occlusal forces, but with clinical examples.\n\nShopova, D., Yordanova, M., & Yordanova, S. (2021). Software Details in Occlusal Splint Creation through 3Shape Design Studio. Open Access Macedonian Journal of Medical Sciences, 9(D), 330-3354. – The treatment plan is very important. The dentist should know the opportunities of digital software, to know how they can help to the patient in every different situation.\n\nShopova, D., Bakova, D., Yordanova, M., & Yordanova, S. (2021). TELEDENTISTRY METHODS IN ORTHODONTICS AND PROSTHETIC DENTISTRY DURING COVID-19 PANDEMIC. KNOWLEDGE-International Journal, 49(4), 667-672. – This article presents digital approach of bruxism treatment again, but this time from pandemic point of view.\n\nShopova, D., Yordanova, M., & Yordanova, S. (2021). 3Shape Digital Design Software in Splints Creation—A Pilot Study. European Journal of Dentistry5. – here it presents the difference in occlusal surfaces in splints, which every dentist has to know, not to leave the choice in dental technicians’ hands.\n\nTaneva, I., Grozdanova-Uzunova, R., & Uzunov, T. (2021, March). Occlusal splints–changes in the muscular activity. In Journal of Physics: Conference Series (Vol. 1859, No. 1, p. 012046). IOP Publishing6. – The condition of the muscles is very important for the successful result of the treatment in bruxism.\n\nTaneva, I., Uzunov, T., & Milanov, N. (2020). Complete digital approach for bruxism management. Problems of Dental Medicine, 46, 18-27. – It presents modern method of bruxism treatment, very similar with the purpose of the reviewed article.\nAll of these articles presented the modern methods of diagnosis and treatment in bruxism cases. They can be find in Google scholar or linked to this report, some of them are uploaded in Scopus or Web of Science. Authors can add and more similar articles to enrich their article. 50 cited articles are too small number.\nLong tables make the results quite difficult. Try to systematize the cited authors more tightly.\nThe discussion is voluminous enough and a sufficient number of authors are compared.\nThe conclusion consists of one short sentence. Authors can submit their opinion on the most suitable app according to them.\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? I cannot comment. A qualified statistician is required.\n\nAre the conclusions drawn adequately supported by the results presented in the review? No",
"responses": [
{
"c_id": "8856",
"date": "03 Oct 2022",
"name": "Byron Velasquez Ron",
"role": "Author Response",
"response": "Good morning dear reviewer, we will gladly make the changes as soon as possible from your valuable comments. Kind regards. Byron"
}
]
},
{
"id": "150308",
"date": "19 Oct 2022",
"name": "Nithin Manchery",
"expertise": [
"Reviewer Expertise General dentistry and older adults."
],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 is an interesting topic of research. A few suggestions for the authors to consider are provided below.\nAbstract.\nResults section - Suggest rephrasing, \" All the authors agree...\" to \" All the included studies found...\"\n\nThe sentence \" The results also show that...the two bruxism...\" is unclear. Please check the sentence.\n\nKindly define/write the words in full and provide abbreviations within brackets before using them elsewhere in the text (e.g., in the discussion section of the abstract: \"AB\").\nIntroduction\nSuggest the knowledge gap could be elaborated a little to strongly justify the rationale for this review (e.g. the data are only partial and little research - what were the findings and how did they compare, are there only the 2 studies so far?).\n\nAlso, to include the potential implications of the findings from this SR.\nMethods\nWere articles restricted only to English? Was this limit applied?\n\nIn the sentence, \"...98 studies were included in the qualitative analysis...\", suggest changing to '98 studies were assessed for eligibility...' for better clarity.\nResults\nConsider explaining the results in detail in word. The basic characteristics, risk of bias, key findings.\n\nWas a meta-analysis considered or avoided due to heterogenicity between the included studies?\nDiscussion\nThe authors constantly compare findings between the 2 types of bruxisms. I suppose this deviates from the main aim - which is only focussed on AB.\n\nAre all the limitations addressed? Bias, strength of evidence.\nConclusion\nPlease add the key findings from the SR and significance of the findings.\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": "8918",
"date": "19 Oct 2022",
"name": "Byron Velasquez Ron",
"role": "Author Response",
"response": "Thanks so much for your commentaries, in this days working with my team, correct the papers and send with corrections. Regards. Byron"
}
]
}
] | 1
|
https://f1000research.com/articles/11-479
|
https://f1000research.com/articles/11-1435/v1
|
05 Dec 22
|
{
"type": "Review",
"title": "The role of open research in improving the standards of evidence synthesis: current challenges and potential solutions in systematic reviews",
"authors": [
"Eirini Martinou",
"Angeliki Angelidi"
],
"abstract": "Systematic reviews (SRs) and meta-analyses (MAs) are the cornerstone of evidence-based medicine and are placed at the top of the level-of-evidence pyramid. To date, there are several methodological resources available from international organizations such as the Cochrane Collaboration that aim to aid researchers in conducting high-quality secondary research and promoting reproducibility, transparency and scientific rigour. Nevertheless, researchers still face challenges in most stages of evidence synthesis. Open research and the FAIR (findability, accessibility, interoperability, and reusability) principles are rising initiatives being increasingly implemented in primary research. However, their beneficial role in secondary research is less emphasized. This article addresses how the challenges commonly faced during evidence synthesis research could be overcome using open research practices and currently available open research tools. Despite the phenomenally simple SR workflow, researchers still find tasks such as framing the SR research question, search strategy development, data extraction, and assessing for bias, challenging. The implementation of FAIR practices, including prospective registration at the PROSPERO database, abiding with the PRISMA guidelines, and making all SR data openly available could have significant benefits in avoiding duplication of effort and reducing research waste while improving the reporting standards of SRs. Additionally, this article highlights the need for further education in open research culture to overcome ethical and motivational barriers in implementing open research practices in evidence synthesis. Finally, in the era of technological breakthroughs, artificial intelligence may eventually be incorporated into the process of SRs and should abide by the FAIR standards for open research.",
"keywords": [
"evidence synthesis",
"open research",
"FAIR principles",
"systematic review"
],
"content": "Introduction\n\nEvidence synthesis refers to any method that identifies, selects, and combines results from multiple studies. It includes reviews (narrative, systematic, rapid, scoping, umbrella) and meta-analyses (MAs).1 Narrative reviews are used widely in evidence synthesis; however, they tend to be descriptive and do not have a standardized methodology or established protocol. On the contrary, a systematic review (SR), according to the Cochrane Collaboration, is defined as a review that identifies, appraises, and synthesizes all the available evidence to answer a specific research question under pre-specified eligibility criteria.2 SRs aim to summarize the available evidence comprehensively and transparently while minimizing bias and enhancing the reliability of the related conclusions.3,4\n\nSince the first publication of a SR by James Lind in 1753, there has been tremendous growth in this field with a more than 20-fold increase in the number of SRs indexed over the last two decades.5 An observational study by Hoffman et al. estimated that approximately 80 SRs were being published per day in the last 20 years and over 200,000 might now be available.5,6 Within the field of clinical practice, SRs and MAs are placed at the top of the level-of-evidence pyramid as they form the basis of clinical practice guidelines (Figure 1).7 SRs are considered the cornerstone of evidence-based medicine; however, their role is expanded in other scientific areas including social sciences and basic science research as they promote the current understanding and facilitate the identification of research gaps.8,9\n\nhttps://www.cebm.ox.ac.uk/resources/levels-of-evidence/oxford-centre-for-evidence-based-medicine-levels-of-evidence-march-2009\n\nCreated with Biorender.com (Academic license ID: TQ24GJ1I5J)\n\nMA: Meta-analysis, RCT: Randomised-controlled Trial, SR: Systematic Review\n\nThe use of a precise and explicit methodology is of paramount importance in conducting SRs and therefore, several methodological resources have been developed to aid researchers in this field.10 To date, several organizations such as the National Institute of Health and Clinical Excellence (NICE) in the UK, the Evidence-based Practice Centre Program in the USA, and the international Campbell and Cochrane Collaborations have been established and are dedicated to developing tools and methods guides for research synthesis and evidence reports (such as SR).11 However, despite the widely available resources, researchers still face challenges in transparency when conducting SRs10\n\nOpen research (also referred to as “open science”) is a term that encompasses the whole research process making the whole research process transparent.12,13 The principles of findability, accessibility, interoperability and reusability (FAIR) were formulated in 2014 and were firstly designed only for data-sharing practices.12,13 However, FAIR principles have been used more broadly in research policies and have become the cornerstone of research data management and stewardship.13 FAIR principles apply not only to data but also to research workflows and protocols used in research studies.12,13 Moreover, the application of FAIR principles could be expanded beyond primary research studies to evidence synthesis research. Our article addresses open research and SRs using the FAIR principles as a basis. Open research is a rapidly evolving shift in research culture to promote transparency and openness in every field of research and is supported by several organizations such as UK Research and Innovation (UKRI), the National Institute of Health Research (NIHR), and the United Nations Educational, Scientific and Cultural Organization (UNESCO). Interestingly, although open research practice is highly promoted, the use of FAIR principles is still lacking in systematic reviews making them challenging to be conducted and their quality questionable.14,15\n\nThis article aims to highlight the current challenges and barriers encountered while conducting SR and suggest potential solutions by emphasizing the importance of open research practices. Moreover, this manuscript describes the role and future perspectives of artificial intelligence in the FAIRness of evidence synthesis. In our article, we first describe the main steps in the SR pipeline. We further review the challenges the researchers may face during these steps of evidence synthesis research and suggest potential solutions by using open research tools and abiding by open research principles. Secondly, we summarize the difficulties and barriers that may impede the full implementation of FAIR principles in SRs, and we propose potential solutions to overcome these challenges. Lastly, we discuss future perspectives regarding the role of using automation tools in SRs within the context of FAIR principles.\n\nSRs as well as MAs are characterized by an explicit and robust methodology which consists of several consecutive steps starting from framing the research question followed by identifying relevant work, appraising the quality of included studies, summarizing the evidence, and interpreting the results.16 However, researchers while conducting an SR, may experience many challenges despite their phenomenally simple workflow.16 FAIR principles refer to the fact that research data should display the following characteristics: data should be registered in a searchable source and assigned a unique identifier, be retrievable ideally through automated procedures, be available to be combined to maximize their value, and lastly be described in detail to facilitate their reuse (Figure 2).12 The movement toward an open research culture that supports the FAIR principles in data sharing may have important implications in the conduct, methodology and reporting of SRs and other evidence synthesis studies.17 Below we discuss the potential obstacles that researchers may often encounter while conducting an SR and how they could overcome these barriers by using open research practices and open relevant tools\n\nFraming the research question\n\nConceptualizing an SR and formulating the research question is the most important step in evidence synthesis studies. A significant challenge that researchers face at this stage is to identify information regarding ongoing SRs addressing the same research question. Ioannides et al. have painted a picture in evidence synthesis research activity where there has been an explosion of SRs and MAs that are often redundant.14 Katsura et al. also suggest that multiple SRs on the same topic represent wasted efforts and they can be confusing and potentially misleading.14,18\n\nInterestingly, until 2011, SRs or meta-analyses were mostly available through search engines such as MEDLINE and EMBASE only after they have been completed and published.14,19 Researchers and healthcare evidence users were facing challenges in finding ongoing SRs as only a limited number of organizations including Cochrane Collaboration contained information related to SRs that were in progress.20 To overcome these challenges and promote FAIRness in evidence synthesis, the International Prospective Register of Systematic Reviews (PROSPERO) was launched in February 2011 as an open-access database where all health-related SRs are eligible for prospective registration.21 Additionally, the status of SRs is regularly updated and therefore researchers and healthcare professionals are able to find SRs which are in progress or are completed but not yet published.19,21 Open-access findability of ongoing SRs provides researchers and commissioners with an important tool to avoid duplication and reduces research waste.22 It is worth highlighting that the ability to promote FAIRness of ongoing SRs through PROSPERO does not only benefit researchers but also is important for clinicians and other healthcare professionals to remain up to date with related ongoing developments in healthcare.22\n\nDespite, the benefits of findability through the PROSPERO open access database and the increase in its use over the years, still, only a fraction of SRs were noted to be registered.14,23 Tawfik et al. by conducting a global survey of 270 authors, reported that only 50% of SRs were officially registered.23 Interestingly, the most common cause was a lack of awareness of the importance of open-access prospective registration of SRs, indicating the need for academic education in open research culture.23 To overcome this observation and encourage PROSPERO registration, the Preferred Reporting Items for Systematic reviews and Meta-Analyses (PRISMA) statement updated its criteria in 2020 for reporting SRs by moving the registration information to a separate checklist section to encourage open research practice within the global research community and promote FAIRness in SRs.24\n\nIdentifying relevant work\n\nTo capture as many relevant citations as possible during an SR, a diligent and thorough search should be conducted.25 An extensive literature search is achieved using multiple electronic databases to identify primary studies according to a priori specified inclusion and exclusion criteria.2,25 Identifying relevant citations is a fundamental step for a reliable SR and includes the development of a meticulous Boolean strategy designed separately for each database. Subsequently, screening steps are performed based on the abstract and full text.2 A survey by Major et al. showed that the design of a search strategy and the performance of an extended literature search were reported to be the most challenging aspects that researchers encountered during an SR.26\n\nThe development of a thorough Boolean strategy algorithm including all the possible vocabulary and synonyms is a relatively complex and time-consuming process which could be overcome by implementing open research principles in SRs through the current available open research tools (PROSPERO).27 A Boolean algorithm which is easily findable and accessible could be reused or modified by several other research teams who wish to conduct an SR on a different subject in the same research field to avoid duplication of research efforts. Interestingly, despite the presence of reporting guidelines such as PRISMA and the need for FAIRness and transparency in reporting the methodology of SRs, several studies have identified that in most SRs, a full Boolean search strategy was missing from the published articles.28–30 To further promote open research practices in SRs, one of the alterations noted in the updated PRISMA 2020 guidelines is the modification of the search term which now recommends that full search strategies for all databases should be reported.24 Additionally, the process of registration in the PROSPERO database requires the submission of the full search strategy; however, making this information publicly available prior to the publication is still optional.21 Recently, preliminary results from the REPRISE study by Page et al. showed that in a random SR sample less than 1% of the included studies reported an analytic search code which was used to generate results, highlighting that despite available open research tools and the revised PRISMA 2020 guidelines, further interventions are needed31,32 The development of common rules across the databases regarding the Boolean strategy design or the presence of easily accessible instructions could potentially aid researchers in tackling the challenging task of analytic vocabulary code design.\n\nThe screening phase of an SR aims to select potentially eligible studies for further assessment based on the prespecified inclusion and exclusion criteria. According to Cochrane guidance, two or more reviewers should independently perform this step, first by screening the title and abstract followed by a full-text assessment of the studies initially included.25 A common challenge that researchers face is the lack of consistent full-text availability leading them to proceed with individual communication with authors requesting full texts of the candidate manuscripts with variable responses.33 Studies have shown that data requests are ignored in up to 41% of cases.33,34 A potential solution to this challenge is proposed to be open-access publishing which is steadily increasing in popularity as articles that are freely accessible are more shared and ultimately benefit the distribution of knowledge.35 However, open-access publishing is associated with increasing article processing charges that place an increased financial burden, especially on unfunded and early-career researchers.36 Although journals and publishers should continue to encourage open-access publishing, we encourage them to consider alternate models of support to reduce the financial barriers that lesser-funded or unfunded researchers face.36 Furthermore, given that institutional affiliation contact details frequently change in a researcher’s career, the availability of research websites such as ResearchGate or other social networking sites for researchers could facilitate communication across the research community and promote accessibility to full text or data of a study. In addition, we believe researchers should be entitled to free access to articles related to the SR they are conducting to promote knowledge and improve the scientific rigour of the study.\n\nData extraction and quality assessment of included studies\n\nThis step encompasses data collection from the included full texts and is accompanied by a quality assessment of the included studies.16 These steps not only require a considerable amount of time and effort but also generate a vast amount of valuable secondary data which could be used and built upon to generate further new knowledge.37\n\nData extraction constitutes a significant portion of an SR. Making all data extracted during SRs openly available could reduce unnecessary duplication of effort from other researchers and promote a standardized format in which data are presented in SRs.38,39 To increase SR data openness, the Systematic Review Data Repository (SRDR) was developed and launched in 2012 by the Brown University Evidence-based Practice Center and funded by Agency for Healthcare Quality and Research (AHRQ).39 This open-access resource aims to serve as an archive for SRs as well as an open-access data extraction tool.38 Saldanha et al. reported that since 2012, SRDR has gathered over 150 SRs and includes publicly available data from more than 15,000 studies covering a variety of health-related research subjects.39 Cochrane collaboration has also made initiatives to support accessibility in SR data and is committed to being fully open access by 2025.40\n\nQuality assessment of included studies and risk of bias reporting are key features of a high-quality quantitative SR and one of the most challenging stages in evidence synthesis.41 Risk of bias assessment requires a thorough judgement of the included studies which may introduce an element of inter-rater variability meaning that the same data may be judged differently between research groups.41 For this challenge, there are several available standardized open tools such as the Cochrane tools (RoB1, RoB2, ROBINS-I), especially in health-related themed SRs which aid researchers in this task.42 Interestingly, although the risk of bias is considered essential according to the PRISMA guidelines, several studies assessing the reporting quality of published SRs showed that risk of bias reporting is performed in less than 50%, raising concerns regarding their quality.43–45 A potential solution to this issue may be provided with the a priori registration of an SR in the PROSPERO open-access database which also requires a detailed study design, search strategy and protocol submission with the aim to minimize bias and promote high methodological standards and reproducibility.19,21,22 Studies by Sideri et al. and Allers et al. reported that SRs which were officially registered in the PROSPERO database appeared to demonstrate higher standards in their methodology and reporting quality.46,47 However, this improvement may not only be attributed to the early accessibility of the registered SRs’ design and protocols but also to the fact that authors may have invested more research time in a high-standard SR research design which may indirectly result in a more robust methodology.46\n\nProviding open access to all aspects of an SR including the risk of bias availability and raw extracted data could provide several benefits including a reduction in duplication of effort, improvement in the quality and efficiency of SRs as well as support for secondary analyses addressing additional research questions.37,48\n\nDespite the undoubtedly significant numerous benefits of SR data sharing, several challenges exist in fully implementing FAIR principles related to motivational and ethical barriers.37 Ethical concerns, including ownership of data, intellectual property of ideas, and recognition of data producers, are some of the barriers related to moral principles that may challenge data sharing and the widespread practice of FAIRness in research studies, including SRs.49 Zuiderwijk et al. performed an SR to investigate what inhibits researchers from openly sharing their data. Their study identified trust as a powerful and impactful driver for FAIR data sharing especially in a pre-publication state.50 Notably, Zuiderwijk et al. characterised fear over data control or recognition of researchers’ efforts on data generation as major trust inhibitors for open data sharing.50 This barrier is widely recognized, and several organizations have highlighted the need for appropriate systems for recognition so that acknowledgements are made as needed.37 For instance, Cochrane collaboration states that authors should “respect and acknowledge the source of the data”. Additionally, the SRDR has adopted data citation with digital object identifiers (DOIs).37 Another practice is the registered reports publication process which is being implemented by an increasing number of journals and publishers where study proposals and protocols are peer-reviewed and pre-accepted before the actual research is undertaken and results are produced.51 Registered reports are an increasingly adopted publication practice as it envisages promoting transparency, reproducibility and openness in data sharing in line with the transparency and openness promotion guidelines.51,52\n\nPersonal drivers and intrinsic motivations have been identified by Zuiderwijk et al. as important factors impacting openness in data sharing.50 Scholars exhibit different character or belief-related traits in terms of being supportive or reserved in open data sharing.50 In an effort towards behavioural and academic culture change, the role of education in an open research culture is of paramount importance.53 Ioannidis et al. highlighted the need for a strong educational curriculum to equip researchers and meta-researchers with the knowledge of best scientific practices to promote FAIRness in meta-research.53 Furthermore, Universities and other academic institutions may consider rewarding meta-researchers who make their analytic code and research data publicly available as proposed by the 6th World Conference on Research Integrity and in line with the Hong Kong principles for assessing researchers to foster trustworthiness, rigour, and transparency.31,54\n\nAlong with the various organizations which promote open research in evidence synthesis and the currently available open research tools, it is worth mentioning the key role that journal publishers and research funders play in enhancing data sharing and its reuse.37 In 2016, the International Committee of Medical Journal Editors (ICMJE) proposed that data from published clinical investigative trials need to be shared to promote transparency and reusability.17 However, to date, no relevant policy exists for evidence synthesis research. Modification of the interface of the existing SR registries to include detailed protocols, search codes and raw data may further promote the reusability of SR/MA-generated data and improve the standards of evidence synthesis reporting.17 Additionally, an increasing number of publications, such as F1000 and Systematic Reviews, provide clearer requirements for datasets to be shared in accordance with the FAIR principles37\n\nUndoubtedly, evidence synthesis research is growing exponentially, therefore the current robust but slow and resource-intensive process of the standard SRs is no longer sustainable.55 Therefore, several methods and tools have been developed through the use of artificial intelligence (AI) in order to aid the automation of SRs.55–57 AI includes machine learning (ML) which utilizes computer algorithms similar to logistic regression and natural language processing (NLP) which analyses a vast number of texts and extracts information.56,58 Both ML and NPL are commonly implemented technologies used in the semi-automated conduction of SRs.58 ML uses statistical predictive methods to calculate the likelihood that an article is relevant and is commonly used during the screening process whereas NPL analyses the semantic meaning and is used during the data extraction step of an SR.59\n\nSeveral automation tools have been developed that aim to execute the time-consuming and labour-intensive tasks of an SR such as search (e.g. Metta), article screening (e.g. Abstrackr), data extraction (e.g. ExaCT) and even automatic generation of PRISMA diagrams (e.g. PRISMA flow diagram generator).55 The significant advance in AI software as well as the desire for openness and FAIRness in meta-research has led to the initiation of the International Collaboration for the Automation of SRs (ICASR).60 In 2015, in the first ICASR meeting, a set of principles were established known as the Vienna principles which recommend the need for a collaborative multidisciplinary effort towards the automation of the evidence synthesis research process while highlighting that every automation technique should abide by the FAIR standards for open research and be freely available.60\n\nDespite the development of several automated platforms for SRs, the complete automation of the evidence synthesis process is not feasible at present, as no current AI tool can replace human judgement.59 Additionally, the benefits of the implementation of AI methods in SRs remain unclear and AI platforms still need to undergo validation and refinement.60 Nevertheless, AI tools are increasingly becoming incorporated into some of the steps of evidence synthesis until a fully automated process might be possible in the near future, (Figure 3). The conduction of evidence synthesis using AI in line with the FAIR principles and the utilization of computer science and health informatics may dramatically alter evidence synthesis research and eventually evidence-based medicine.56,59,61\n\nSources Beller et al. and John Hopkins University of Medicine (https://browse.welch.jhmi.edu/sr-methods/sr-process). Created with Biorender.com (Academic license ID: KV24GJF9KY).\n\nAI: Artificial intelligence, SRDR: Systematic Review Data Repository, SR: systematic review\n\n\nConclusion\n\nIn conclusion, this review has highlighted the need for a transparent approach in evidence synthesis research in which implementing FAIR principles play a crucial role in tackling methodological challenges as well as improving the reporting standards of SRs. These may have broader benefits related to public health issues as data extracted from high-quality SRs could accelerate the conduction of rapid evidence-based reviews to produce information promptly, promote research dissemination and engage effective strategies, especially in global health crises such as major outbreaks or pandemics.37,62 Since existing SRs are often used by rapid reviews, making SR data as openly available as possible could provide vital answers at high standards and in a timely fashion for governments and policy makers in case of public health threats.63\n\nFinally, educating evidence synthesis researchers to follow the FAIR guidelines is of paramount importance for an academic cultural shift towards open research. AI may play an important role in the future of SRs and may aid in a more standardized and open evidence synthesis practice.\n\nNo data are associated with this article\n\n\nAuthor contributions\n\nConceptualisation: EM, AA; Project administration: EM, AA; Writing of original draft: EM, AA; Visualisation: EM, AA; Review and Editing: EM, AA",
"appendix": "Acknowledgements\n\nThe authors would like to thank Dr Daniele Kurtin and Professor Emily Farran, University of Surrey for their contribution during the editing process of the article (written consent was obtained)\n\n\nReferences\n\nGough D, Davies P, Jamtvedt G, et al.: Evidence Synthesis International (ESI): Position Statement. Syst. Rev. 2020 Dec 10; 9(1): 155. PubMed Abstract | Publisher Full Text | Free Full Text\n\nHiggins JPT, Thomas J, Chandler J, et al.: Cochrane Handbook for Systematic Reviews of Interventions version 6.3 (updated February 2022). Cochrane;2022. [cited 2022 Jul 28].Reference Source\n\nMurad H, Asi N, Alsawas M, et al.: New evidence pyramid.2016; 21.Reference Source\n\nHorvath AR, Pewsner D: Systematic reviews in laboratory medicine: principles, processes and practical considerations. Clin. Chim. Acta. 2004 Apr 1; 342(1–2): 23–39. PubMed Abstract | Publisher Full Text\n\nClarke M, Chalmers I: Reflections on the history of systematic reviews. BMJ. 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PubMed Abstract | Publisher Full Text | Free Full Text\n\nVervoort D, Ma X, Bookholane H: Equitable Open Access Publishing: Changing the Financial Power Dynamics in Academia. Glob Health Sci Pract. 2021 Dec 31; 9(4): 733–736. PubMed Abstract | Publisher Full Text | Free Full Text\n\nYoong SL, Turon H, Grady A, et al.: The benefits of data sharing and ensuring open sources of systematic review data. J Public Health (Bangkok). 2022 Mar 14; PubMed Abstract | Publisher Full Text\n\nIp S, Hadar N, Keefe S, et al.: A Web-based archive of systematic review data.2012.Reference Source\n\nSaldanha IJ, Smith BT, Ntzani E, et al.: The Systematic Review Data Repository (SRDR): descriptive characteristics of publicly available data and opportunities for research. Syst. Rev. 2019 Dec 20; 8(1): 334. PubMed Abstract | Publisher Full Text | Free Full Text\n\nCochrane Community: Strategy for Change: 2021-2023.[cited 2022 Aug 2].Reference Source\n\nFrampton G, Whaley P, Bennett M, et al.: Principles and framework for assessing the risk of bias for studies included in comparative quantitative environmental systematic reviews. Environ Evid. 2022 Dec 29; 11(1): 12. Publisher Full Text\n\nJardim PSJ, Rose CJ, Ames HM, et al.: Automating risk of bias assessment in systematic reviews: a real-time mixed methods comparison of human researchers to a machine learning system. BMC Med. Res. Methodol. 2022 Dec 8; 22(1): 167. PubMed Abstract | Publisher Full Text | Free Full Text\n\nKatikireddi SV, Egan M, Petticrew M: How do systematic reviews incorporate risk of bias assessments into the synthesis of evidence? A methodological study. J Epidemiol Community Health (1978). 2015 Feb; 69(2): 189–195. PubMed Abstract | Publisher Full Text | Free Full Text\n\nWu IX, Wang H, Zhu L, et al.: Methodological quality of systematic reviews on interventions for osteoarthritis: a cross-sectional study. Ther Adv Musculoskelet Dis. 2020 Jan 23; 12: 1759720X2095996. PubMed Abstract | Publisher Full Text | Free Full Text\n\nPage MJ, Moher D: Evaluations of the uptake and impact of the Preferred Reporting Items for Systematic reviews and Meta-Analyses (PRISMA) Statement and extensions: a scoping review.\n\nSideri S, Papageorgiou SN, Eliades T: Registration in the international prospective register of systematic reviews (PROSPERO) of systematic review protocols was associated with increased review quality. J. Clin. Epidemiol. 2018 Aug; 100: 103–110. Publisher Full Text\n\nAllers K, Hoffmann F, Mathes T, et al.: Systematic reviews with published protocols compared to those without: more effort, older search. J. Clin. Epidemiol. 2018 Mar; 95: 102–110. PubMed Abstract | Publisher Full Text\n\nWolfenden L, Grimshaw J, Williams CM, et al.: Time to consider sharing data extracted from trials included in systematic reviews. Syst. Rev. 2016 Dec 3; 5(1): 185. PubMed Abstract | Publisher Full Text | Free Full Text\n\nKalkman S, Mostert M, Gerlinger C, et al.: Responsible data sharing in international health research: a systematic review of principles and norms. BMC Med. Ethics. 2019 Dec 28; 20(1): 21. PubMed Abstract | Publisher Full Text | Free Full Text\n\nZuiderwijk A, Shinde R, Jeng W: What drives and inhibits researchers to share and use open research data? A systematic literature review to analyze factors influencing open research data adoption. PLoS One. 2020 Sep 18; 15(9): e0239283. PubMed Abstract | Publisher Full Text | Free Full Text\n\nChambers CD, Tzavella L: The past, present and future of Registered Reports. Nat. Hum. Behav. 2022 Jan 15; 6(1): 29–42. PubMed Abstract | Publisher Full Text\n\nNosek BA, Alter G, Banks GC, et al.: Promoting an open research culture. Science (1979). 2015 Jun 26; 348(6242): 1422–1425. Publisher Full Text\n\nIoannidis JPA, Fanelli D, Dunne DD, et al.: Meta-research: Evaluation and Improvement of Research Methods and Practices. PLoS Biol. 2015 Oct 2; 13(10): e1002264. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMoher D, Bouter L, Kleinert S, et al.: The Hong Kong Principles for assessing researchers: Fostering research integrity. PLoS Biol. 2020 Jul 16; 18(7): e3000737. PubMed Abstract | Publisher Full Text | Free Full Text\n\nTsafnat G, Glasziou P, Choong MK, et al.: Systematic review automation technologies. Systematic review automation technologies. Syst Rev. 2014 Dec 9; 3(1): 74. Publisher Full Text\n\nBlaizot A, Veettil SK, Saidoung P, et al.: Using artificial intelligence methods for systematic review in health sciences: A systematic review. Res. Synth. Methods. 2022 May 28; 13(3): 353–362. PubMed Abstract | Publisher Full Text\n\nBozada T, Borden J, Workman J, et al.: Sysrev: A FAIR Platform for Data Curation and Systematic Evidence Review. Front Artif Intell. 2021 Aug 5; 4. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMarshall IJ, Wallace BC: Toward systematic review automation: a practical guide to using machine learning tools in research synthesis. Syst. Rev. 2019 Dec 11; 8(1): 163. PubMed Abstract | Publisher Full Text | Free Full Text\n\nValentin CD: Systematic Review Writing by Artificial Intelligence: Can Artificial Intelligence Replace Humans? J. Musculoskelet. Disord. Treat. 2022 Jun 30; 8(1). Publisher Full Text\n\nBeller E, Clark J, Tsafnat G, et al.: Making progress with the automation of systematic reviews: principles of the International Collaboration for the Automation of Systematic Reviews (ICASR). Syst. Rev. 2018 Dec 19; 7(1): 77. 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}
|
[
{
"id": "160765",
"date": "06 Feb 2023",
"name": "Jennifer Hunter",
"expertise": [
"Reviewer Expertise Systematic reviews and other evidence synthesis methods"
],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 discusses some of the challenges with conducting systematic reviews (SRs) and argues for the use of open research practices based on the FAIR principles (findability, accessibility, interoperability, and reusability) to help improve the standards of SRs and reduce research waste. The article also highlights the need for education in open research culture and the emerging role of artificial intelligence.\nThe following revisions are suggested.\nDescribing the SR workflow as \"phenomenally simple\" in the abstract and manuscript suggests that the process is straightforward and uncomplicated, yet researchers still face challenges in various stages of the process. Suggest replacing with something like, “relatively straightforward workflow”.\n\nThe abstract, manuscript and Figure 1 conflate a meta-analysis (MA) with a systematic review (SR). A MA is a statistical tool. A SR is a study that aims to identify and synthesize all the available evidence. On its own, a MA it is not Level I evidence, as these levels of evidence are referring to study designs and their quality, not the analytical methods used to generate the results. Additionally, MAs can be used to analyse data in other types of non-systematic studies. Recommend editing throughout to address this.\n\nFigure 1 needs further work. It looks like the authors have attempted to amalgamate the five columns in the cited table (Oxford Centre for Evidence-Based Medicine: Levels of Evidence (March 2009) into one column. There are numerous discrepancies such as SRs of cohort studies being listed as both Level Ia and Level II. The focus of this article is evidence synthesis and SRs, not evidence hierarchies and decision making, so it might be more useful to replace this figure with a diagram or table that summarises the different types of SRs of primary and secondary studies (e.g., the types of Cochrane reviews https://www.cochranelibrary.com/about/about-cochrane-reviews)\n\nRecommend adding the Joanna Brigs Institute (JBI) to the list of organizations that “are dedicated to developing tools and methods guides for research synthesis and evidence reports”. https://doi.org/10.1016/j.jclinepi.2022.04.008\n\nRecommend listing some of the other open access options for registering SR protocols. This is particularly relevant for other types of evidence synthesis (e.g. scoping review) that are not accepted by PROPSERO.\n\nPage 6. Second paragraph has a minor grammatical error “an SR” should be “a SR”\n\nThe conclusion would benefit from more work – it jumps around and lacks flow.\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? Yes",
"responses": []
},
{
"id": "169522",
"date": "27 Apr 2023",
"name": "Farhad Shokraneh",
"expertise": [
"Reviewer Expertise Evidence Synthesis",
"Systematic Reviews"
],
"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 topic of this review is interdisciplinary (evidence synthesis and open science), which is considered an ongoing interest for many in the evidence synthesis community. I appreciate both authors' interest in the topic and encourage them to continue studying and practising open research in the evidence synthesis context; however, as an independent peer, I have to be critical of the manuscript. I hope my criticism could constructively improve the quality of the manuscript without being taken as personally.\nExpertise in the Field of the Review A review of such importance could have been co-authored by experts (researchers, practitioners, and policymakers) on both or at least one of these topics and submitted to the most relevant journal for visibility to the relevant community members. Lacking such expertise becomes clearer as one reads that the SR workflow is \"phenomenally simple\" in the abstract. I wondered if the authors considered or will consider inviting experts (from the list of cited references) to co-author the next version with them, as it happens that because of time constraints, some authors rush towards submission and make the main effort in the revised version.\nDefinition of Evidence Synthesis The authors need to present a definition of evidence synthesis and/or systematic review that fits their review's purpose. The use of components of systematic reviewing as part of evidence synthesis is very popular and a source of confusion. For example, if a team reports the keywords used for searching for their review, should we call it a systematic review? What if they only report a search strategy without following the other steps or don't conduct a risk of bias assessment?\nPurpose The writing style could be more coherent for a review article. This manuscript could be perceived as the first version of a self-reflective work when someone new to the field studies papers and tries to write an introductory chapter for a dissertation or coursework. In the current format, the review serves a purpose other than scientific communications with the community through publishing a review in an academic journal. I hope the authors find an acceptable gap in the literature to fill by publishing this review.\nContribution to the Field This review does not add to the already known, documented, and updated knowledge in this field and does not summarise or enrich the existing knowledge. While the authors have selectively and briefly used some of the widely known literature (Tsafnat et al. 2014; Ross 2016; Wolfenden et al. 2016; ICASR and Vienna Principles), they have missed some of the most important literature in the field (Haddaway 2018 1; Metzendorf et al. 2022 2; Shokraneh et al. 2018 3; Shokraneh 2019 4; Uttley et al. 2023 5). It should also be noted how some previous experts have published their works as commentary, letters, and editorials, not necessarily as reviews.\nInternal and External Facilitators of Open Science The authors have focused on highlighting the role of open science from an internal perspective, only focusing on what systematic reviewers can do without discussing the external barriers such as blockage of open science by publishers through copyright, open access article processing charge that stops unfunded or less-funded research from becoming visible, and publishing/trapping information/data in PDF format which is one of the worst enemies of openness. Other external barriers are the primary researchers not practising open science principles that, make the data for systematic reviews and automation unavailable.\nTechnical Comments\nSR workflow might look \"phenomenally simple\" from a non-expert point of view, but it's common knowledge in the expert community that no systematic review process could be called simple.\n\nMEDLINE and Embase are not search engines; however, they have search interfaces that may be called search engines for simplicity. These sources are technically known as bibliographic medical databases.\n\nPROSPERO is only one of the available platforms for registering systematic reviews, and it does not accept all types of reviews (or systematic reviews).\n\nPRISMA 2009 mandated reporting search strategy for only one database, contributing to waste and a barrier to openness and reproducibility. PRISMA 2020 and PRISMA-Search mandate reporting all search strategies for all sources.\n\nChallenges of systematic searching and possible solutions have already been reported, and the authors have cited them (MacFarlane et al. 2022). Selectively repeating them could lead to highlighting only some of the issues.\n\nsearchRxiv is the optional open registry for reporting search strategies, not PROSPERO. It is possible to register a review in PROSPERO without a search strategy.\n\nThe authors have cited the old strategy for Cochrane. In \"Our next strategy: proposed new strategic framework to 2025\", Cochrane will try to \"improve access to Cochrane data for research users\". While it is not Cochrane's data to hold, Cochrane shares data upon request and does not make it publicly and openly available in line with FAIR or CC.\n\nA problem with systematic reviews is that they are time-consuming and resource intensive; however, beginners see reviews as an easy way to have a publication in their resume. As a result, many start the review, but they can't finish it. Many conduct reviews to publish rather than answer a real-world question or problem. Some look at systematic review workflow as phenomenally simple so they can start and finish a review without funding (time, expertise, or resources). If they finish, the result usually suffers from low quality or answers a question with no or low priority, so it does not serve the science, policy, or practice.\n\nSome references have been cited in a rush and must be completed and corrected.\nAutomation as Solution\nThe discussion on automation is a brief and uninformative summary of what has been presented systematically and coherently in previous works. The most important source of automation tools (Systematic Review Toolbox) has yet to be mentioned or cited.\n\nThe authors have only cited access to the full text as the main problem for the identification stage, missing the aforementioned external barriers. Most importantly, the main ML help we have received so far is using ML for screening the search results. While progressing on other SR steps, ML/AI has a long way to go.\n\nHaving a standard list of data points (metadata) for systematic reviews is almost impossible. Each systematic review is unique and requires setting up data extraction and entry forms with a different number of data points and metadata.\n\nAutomation has little or no success in data extraction because of a lack of standards in reporting primary studies, using PDF rather than HTML as the primary format, and a lack of standards in analysing or reporting the analysed or measured data. Some publishers also hold copyright/ownership of data, now allowing unrestricted use. Many researchers find data sharing an extra task to their already overloaded schedule, especially if they have not received any training. If not enforced, they avoid sharing.\n\nIt should be considered that any solutions today can only help to resolve future problems. Retrospective trapped science will remain in commercial publishers' prisons.\n\nThe risk of bias assessment as part of SR depends on the definition of SR and field and the availability of reliable and valid tools for the included study designs. Automation can only help some reviews as human judgement is usually required.\n\nParadoxically, instead of presenting the tools and sources compliant with open science principles, the authors listed the tools that do not follow such principles in the text and figure.\n\nFinal Words I appreciate the authors' interest in the field to study and summarise some important literature for this introductory piece. I see no addition to the existing knowledge and even supplying incomplete and misleading information that would confuse the readers. Since the authors advocate avoiding duplication, reducing waste, and open science, I'd request that the authors write the second version of the review in line with these three recommendations. This article requires at least 6 to 12 months of part-time work and the involvement of at least two experts in the authorship team to become an impactful work with new insights. I will gladly allocate another six hours to reading and commenting on the next version.\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? Yes\n\nAre the conclusions drawn appropriate in the context of the current research literature? Partly",
"responses": []
}
] | 1
|
https://f1000research.com/articles/11-1435
|
https://f1000research.com/articles/11-1433/v1
|
05 Dec 22
|
{
"type": "Research Article",
"title": "Interrogation of an ovine serum peptide spectral library to annotate ambiguous clinicopathological biomarkers using data-independent acquisition",
"authors": [
"Saul Chemonges"
],
"abstract": "Background: The use of data-independent data acquisition mass spectrometry (DIA-MS) on biological samples from domestic animals is still uncommon. Here, sequential window acquisition of all theoretical mass spectra (SWATH-MS) – a variant of DIA-MS was used to analyse serum peptides of healthy sheep as compared with serum of sick sheep by interrogating a novel peptide spectral library (PSL). This approach enabled the detection and annotation of a wide range of proteins, than conventional clinical pathology protein assays. Methods: Serum samples from healthy sheep were obtained from a commercial source and normalised to represent a healthy sheep proteome background and then compared with serum samples of sheep suffering from a range of naturally-acquired illnesses submitted to The University of Queensland, Australia. Purified tryptic peptides were subjected to liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) on a quadrupole time-of-flight instrument (TripleTOF 5600+, SCIEX) set in a cyclic data-independent acquisition (DIA) mode using a generic (SWATH™, SCIEX) acquisition method. Data were processed using PeakView® v2.2 software with SWATH™ Acquisition MicroApp 2.0 (SCIEX) and MarkerView™ v1.3 software (SCIEX) pipeline to generate protein lists for downstream gene ontology annotation and pathway analysis of identified proteins. Results: There were distinct differences in peptide chromatographic features of sick sheep samples compared to those from healthy sheep. Healthy and sick sheep serum samples yielded 335 and 236 protein identifications (IDs), respectively. There were 96 protein IDs unique to sick sheep serum. A total of 431 protein IDs were annotated by combining healthy control and sick sheep protein IDs. Conclusions: SWATH analysis successfully aided in the detection some established clinicopathological serum biochemical analytes. This approach enabled the distinction of protein profiles of sick sheep samples from a healthy control sample, thereby providing a promising future perspective for the application of SWATH analysis in veterinary clinical use.",
"keywords": [
"Veterinary SWATH analysis",
"peptide spectral library",
"sheep serum proteomics",
"TICs",
"annotation of proteins in serum",
"nanoLC-nanoESI-MS/MS",
"sick sheep",
"healthy sheep"
],
"content": "List of abbreviations\n\nACN: Acetonitrile\n\nALP: Alkaline phosphatase\n\nAST: Aspartate aminotransferase\n\nATP: Adenosine triphosphate\n\nAUC: Area under curve\n\nBCA: Bicinchoninic acid assay\n\nCARF: Central Analytical Facility\n\nCCKR: Cholecystokinin receptors\n\nCK: Creatine Kinase\n\nDIA: Data independent acquisition\n\nDTT: Dithiothreitol\n\nEBI: European Bioinformatics Institute\n\nEGF: Epidermal growth factor\n\nEMBL: European Molecular Biology Laboratory\n\nFA: Formic acid\n\nFGF: Fibroblast growth factor\n\nGGT: Gamma-glutamyl transferase\n\nGO: Gene ontology\n\nIAM: Iodoacetamide\n\nID: Identification\n\nLC: Liquid chromatography\n\nMS/MS: tandem mass spectrometry\n\nMS: Mass spectrometry\n\nnanoLC-nanoESI-MS/MS: nano liquid chromatography nano electrospray ionisation tandem mass spectrometry\n\nPANTHER: Protein ANalysis THrough Evolutionary Relationships\n\nPCA: Principal component analysis\n\nPRIDE: Proteomics identifications\n\nPSL: Peptide spectral library\n\nQUT: Queensland University of Technology\n\nRT: Room temperature\n\nSWATH: Sequential acquisition of all theoretical fragrant mass spectra\n\nTEMED: Tetramethylethylenediamine\n\nTFA: Trifluoroacetic acid\n\nTOF: Time-of-flight\n\nTP: Total protein\n\nUQ: The University of Queensland\n\n\nIntroduction\n\nUntil now, the use of sequential window acquisition of all theoretical fragment mass spectra (SWATH-MS) analysis1 to complement traditional shotgun MS-based proteomics2 on samples from species of veterinary importance has been uncommon. Only a limited number of published reports have applied this approach on animal experimental subjects, for example in the quantification of hepatic proteins of chicken exposed to heat stress,3 analysis of seminal plasma protein of pigs,4 identification of proteins involved in nutritional stress in goats,5 quantification of protein alterations in plasma of acutely endotoxaemic sheep,6 and detecting proteins in peptide spectral libraries of bovids.7 Otherwise, much of the work in this area has been centred upon in-vitro studies on cell lysates8–11 and body fluids obtained from mice.12 Despite its promising nature in protein analysis, SWATH-MS analysis remains largely untested on actual clinical samples from veterinary patients.\n\nIn this report, SWATH-MS was used to analyse serum samples obtained from sheep suffering from a range of naturally-acquired illnesses and compared with samples from healthy sheep, by interrogating a novel peptide spectral library (PSL) constructed from the ovine circulating acellular proteome.6,13 The sick sheep serum samples were of actual clinical cases submitted to The University of Queensland (UQ) (Gatton, Australia). Serum samples from twenty healthy sheep were obtained from a commercial source (Serum Australis Pty Ltd.) and normalised to represent an analytical normal proteome background. Tryptic peptides were purified by StageTip technique,14,15 prior to nano-liquid chromatography nano-electrospray ionisation tandem mass spectrometry (nanoLC-nanoESI-MS/MS) in a cyclic data-independent acquisition (DIA) mode, using a generic SWATH-MS acquisition method (SWATH™, SCIEX). The acquired data were processed in PeakView® software with SWATH™ Acquisition MicroApp 2.0 (SCIEX) pipeline to generate protein lists for downstream gene ontology annotation and pathway analysis of identified proteins.\n\nThe use of SWATH-MS analysis enabled protein profiles of individual sick sheep to be differentiated as compared to the normalised healthy sheep serum aliquots. This approach detected some established clinical biochemical analytes and possibly other candidate disease markers. These observations suggest that it is potentially feasible to use SWATH-MS in conjunction with the novel PSL to determine relative protein alterations in clinical samples from veterinary patients in future.\n\n\nMethods\n\nThe experiments included in this study were conducted in accordance with the Australian Code of Practice for the Care and Use of Animals for Scientific Purposes.16 Animal tissue samples for the generation of the PSL were acquired following approval from Queensland University of Technology (QUT) Confirmation number: 1400000591, QV reference number: 46375 and Ethics number TRIM 10/8428 issued to Serum Australis Pty Ltd (http://www.serumaustralis.com.au), for bleeding live sheep and supply of blood products. Tissue from live animal experiments had animal ethics approval obtained from the University Animal Ethics Committee of QUT (Reference No. 0800000555), which was ratified by The University of Queensland (UQ).\n\nEx-diagnostic serum samples of seven sheep that presented with a range of clinical manifestations each, were opportunistically obtained from UQ laboratory archive for analysis. The accompanying clinical details of these sheep are presented in Additional file 1.48 In this set of experiments, the individual code-identified samples from each sheep (SCi502, SCi503, SCi504, SCi507, SCi508, SCi509 and SCi510), a pooled sample of all of them (SCi511) and a pooled sample of aliquots from 20 healthy sheep obtained from Serum Australis Pty Ltd (SA) (SCi512) as the normalised analytical control for the experiments, were prepared and subjected to proteomic analysis using the SWATH-MS (SCIEX) analysis pipeline (Figure 1).\n\nSerum samples derived from seven sick sheep (SWATH Sample ID: SCi502, SCi503, SCi504, SCi507, SCi508, SCi509 and SCi510), a pooled sample from all the sick sheep (pooled sick) (SWATH Sample ID SCi511) and a normalised sample pooled from 20 healthy sheep (pooled normal) (SWATH Sample ID SCi512). The acquired SWATH data from each sample were processed in PeakView® v2.2 software with SWATH™ Acquisition MicroApp 2.0 (SCIEX) – SWATH pipeline. Data were exported into MarkerView™ v1.3 software (SCIEX) for statistical analysis protein list generation. Protein lists were exported to spreadsheet (Microsoft® Excel™), which facilitated comparative analysis using a Venn diagram (BioVenn software).\n\nSamples were prepared as in a previously described method.13 In brief, frozen sheep serum samples were thawed and kept under refrigeration conditions with an added protease inhibitor (cOmplete™, Roche) according to the manufacturer’s instructions. Each sample was agitated vigorously for half a minute before centrifuging at 13,000 g for 20 minutes. Only the supernatant was retained for downstream analysis, after determining the protein concentration with bicinchoninic acid (BCA) protein assay method (BCA Protein Assay Kit, Pierce™) and a spectrophotometer (NanoDrop 2000; Thermo Scientific).\n\nProteins in the sample supernatants were precipitated by adding 4 × (v:v %) of cold acetone (20 °C) and then incubated at this temperature for 16 h, followed by centrifugation at 4,000 g for 2 minutes. The pellet was then retained and washed using 1 mL of cold acetone by agitating vigorously to break the pellet. The suspension was then centrifuged at 4,000 g for 5 min at 4 °C and sediment was retained. This washing procedure was performed once more. The pellet was dissolved in aqueous fresh 8 M urea in 25 mM ammonium bicarbonate (NH4HCO3) and then centrifuged at 4,000 g for 5 min at 4 °C. The protein concentration of the supernatant was then determined by BCA protein assay.\n\nWhile still under refrigeration conditions, sample aliquots comprising 100 μg of protein were reduced with 20 mM DTT (titrated to 5 mM final concentration) and then incubated for 1h at room temperature (RT), followed by alkylation with 55 mM iodoacetamide (IAM) (resulting in 14 mM final concentration) and incubated again for 20 min in the dark at RT. Alkylation was suppressed using dithiothreitol (DTT) prior to further incubation for 5 min in the dark after which the solution was diluted with 25mM NH4HCO3 buffer followed by titrating aqueous 70 mM CaCl2 into the samples (aiming at 10 mM final concentration). Trypsin (Promega) was added at a ratio of 1:50 (enzyme to protein concentration) and the contents were incubated for 16 h at 37 °C with gentle shaking and then cooled to RT. Protein digestion was curtailed using 10% trifluoroacetic acid (TFA) before vacuum drying the contents. Peptides were then dissolved in aqueous 0.1% TFA/2% acetone nitrile (ACN) and routinely desalted using octadecyl carbon chain (C18) pipette tips (ZipTip® Pipette Tips, Millipore), vacuum dried, reconstituted in 10 uL of aqueous 2% ACN/0.1% FA for nanoLC-nanoESI-MS/MS analysis.\n\n\nnanoLC-nanoESI-MS/MS analysis\n\nQuantities of about 400 ng – 1 μg of peptides per sample were subjected to reversed-phase chromatography setup in a trap and elute arrangement across a 90 min gradient at 40 °C. Two mobile phases A and B were used, prepared from aqueous 0.1% formic acid (FA) and ACN/0.1% FA, respectively on a nanoLC-nanoESI-MS/MS system (TripleTOF® 5600+ instrument (SCIEX)).\n\nSpectral data of purified eluted peptides were extracted by cyclic DIA using a generic SWATH™ acquisition method described elsewhere.1,3 The mass spectrometer was operated using 0.1 s for the survey MS run, followed by tandem mass spectrometry on all precursors in a recurring manner with an accumulation time of 0.1 s per SWATH window across 36 windowsl, each 26 m/z units wide for total cycle duration of 3.75 s in order to sample at least 6 data points for each chromatographic peak per peptide to ensure improved quantification precision.\n\nThe proprietary raw data files (.wiff format) were concurrently imported into PeakView® v.2.2 software with integrated SWATH™ Acquisition MicroApp 2.0.1 (SCIEX) for spectral alignment and targeted data extraction from the PSL to be interrogated. The PSL was assembled from hundreds of samples derived from serum and plasma of sick and healthy sheep that had hitherto been analysed by nanoLC-nanoESI-MS/MS by data dependent acquisition (DDA) on the same instrument as described in earlier.6,13 Extracted ion chromatograms (XICs) from MS/MS spectra for targeted peptides were generated by the SWATH™ Acquisition MicroApp 2.0.1, by assimilating peak areas from the SWATH™ data files as detailed in the vendor’s technical note (http://tinyurl.com/j6w83pu).\n\nThe parameters for SWATH™ data extraction were set as follows: five peptides per protein, five transitions per peptide, peptide confidence level of >95%, exclude shared peptides, and extracted ion chromatograph (XIC) width of 50 ppm. Processed data were exported as quantitative output for the peak area under the intensity curve for individual ions, the summed intensity of individual ions for a given peptide, and the summed intensity of peptides for a given protein in .txt format. Data were then statistically analysed using MarkerView™ software (SCIEX). Data were then exported in.xlxs format (Microsoft® Excel™) into spreadsheet for inspection and comparative visual analysis using BioVenn software.17\n\nThere are other open-access alternative software that can be used to for analysis of the data used in this article, for example the Trans-Proteomic Pipeline (TPP),18 Skyline19 and OpenSWATH.20,21\n\nThe mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium22 via the PRIDE23 partner repository with the dataset identifiers PXD005002 and PXD005077.\n\n\nResults\n\nThe total ion chromatograms of each of the analysed peptides derived from serum samples of sick sheep were processed in PeakView® software and compared with that of healthy sheep (Additional file 248). A total of 335 proteins (Additional file 3(a)48) were identified in the healthy sheep sample pool (SCi512) that was derived from aliquots of 20 sheep, representing the normal proteome background. Protein identifications (IDs) from individual samples of sick sheep were compared to the IDs of SCi512 to reveal shared and unshared overlaps in protein IDs, and total combined protein IDs in each individual clinical case experiment as shown in Figure 2A. A comparison of protein IDs from pooled normal sheep serum (SCi512), pooled sick sheep (SCi511) and those unique to sick sheep only is presented Figure 2B. There were proteins that were unique to each of the sick sheep samples, as compared to healthy sheep pool. These unique proteins numbered 55, 95, 61, 54, 80, 61, 30, and 70 for SCi502, SCi503, SCi504, SCi507, SCi508, SCi509, SCi510, and SCi511, respectively. The UniProtKB entries for the individual proteins unique to each sick sheep sample are presented in Additional file 3 (b), (c), (d), (e), (f), (g), (h) and (i)48, respectively.\n\nA - Shows the number of protein IDs obtained using SWATH™ processing of individual serum samples SCi504, SCi503, SCi504, SCi507, SCi508, SCi509 and SCi510 from sick sheep, and a pooled sample from all the preceding samples (SCi511), that were compared with a pooled serum sample from healthy sheep (SCi512). B - Illustrates a composite analysis of all the samples that yielded 431 protein IDs, 335 of these IDs were attributed to SCi512, 210 IDs were from the pooled sick sheep sample (SCi511) and 96 IDs were exclusive to sick sheep serum only. The 26 protein IDs that were not detected in SCi511 were identified by analysing proteins that were exclusive to sick sheep (sick sheep only fractions) of the individual samples in A.\n\nThe UniProtKB entries for the 195 proteins that were identified as being unique to the healthy sheep sample pool (i.e. proteins that were not detected in sick sheep samples illustrated in Figure 2B), are listed in Additional file 3 (j).48 Also listed, are the 96 protein IDs (Additional file 3 k48) that were unique to sick sheep and the 26 protein IDs (Additional file 3 (l)48) that were not detected in the sick sheep pool (SCi511) compiled from protein IDs exclusive to the sick sheep samples. The UniProtKB entries for all of the 431 proteins identified in the entire workflow comprising of serum samples from sick and healthy sheep in Figure 2B are listed in Additional file 3 (m).48\n\nThere was differential abundance in proteins identified across all the analysed samples, including protein IDs from healthy sheep. The relative intensities of the top 10 most abundant protein IDs in healthy sheep serum pool in descending order, alongside protein IDs from the seven individual sick sheep samples (SCi502, SCi503, SCi504, SCi507, SCi508, SCi509 and SCi510), and the pooled sample from sick sheep (SCi511) are illustrated in Figure 3. The relative abundance of many proteins was variable between the different samples as presented in Additional file 4.48 There were notable differences in protein intensities between the healthy sheep serum pool and the sick sheep sample pool as shown in Figure 4.\n\nProtein intensities from a pooled sample of healthy sheep (SCi512) in descending order were compared with intensities from sick sheep samples (SCi502, SCi503, SCi504, SCi507, SCi508, SCi509, SCi510 and SCi511 (pooled sick)) and corresponding proteins.\n\nA – Shows the relative summed protein intensities of all proteins identified by SWATH-MS analysis in a pooled sample from healthy sheep (SCi512) as compared with a pooled sample (SCi511) derived from 7 samples (SCi502, SCi503, SCi504, SCi507, SCi508, SCi509 and SCi510) from sick sheep. B – Shows the quantitative comparison between the two samples representing healthy sheep and sick sheep was possible, however only 140 identified proteins were common between SCi512 and SCi511. In (B), taking the protein intensities calibration plot from SCi512 as the normalised control (or benchmark), protein IDs in sample SCi511 falling above the blue line were considered upregulated, whilst those below this line were designated as downregulated. Note the wide dynamic range in the intensities of some proteins in both (A) and (B).\n\nOf the 335 proteins identified in the healthy sheep serum pool, the ten most abundant proteins (see Figure 2B and Additional file 3(a)48) in descending order were W5PU57 (Nuclear envelope pore membrane protein POM 121C), W5PBH6 (RAD54 like 2), W5QGI6 (Exonuclease 3'-5' domain containing 1), W5P860 (Dual-specificity kinase), W5Q2U7 (Plectin), O02762 (Apolipoprotein A1), W5Q7I2 (I Ig-like domain-containing protein), C5IS96 (Lecithin-cholesterol acyltransferase), W5PI54 (Rabphilin 3A) and W5P8F9 (BPI1 domain-containing protein). The ten least abundant proteins in this healthy sheep sample pool were W5P0B2 (LDL receptor related protein 2), W5QBV7 (CD44 antigen), W5PAS6 (EvC ciliary complex subunit 2), W5QCV8 (Cadherin EGF LAG seven-pass G-type receptor 2), W5P640 (Lamin A/C), W5PQ96 (Ciliary rootlet coiled-coil, rootletin), W5P7B1 (Sirtuin 2), W5QGV5 (Dedicator of cytokinesis 10), W5QGX2 (Spectrin beta, non-erythrocytic 5) and A2P2G4 (VH region).\n\nOf the 210 proteins identified in the sick sheep serum sample pool (SCi511)(Figure 2B), the ten most abundant proteins in descending order were W5QIV1 (Protein S100), W5QHZ8 (Ig-like domain-containing protein), W5PGA9 (Nicastrin), W5PI92 (Apolipoprotein C-IV), W5NV45 (Hephaestin like 1), A2P2I4 (VH region), W5P640 (Lamin A/C), B0FZL9 (Pre-mRNA splicing factor SRP20-like protein), W5PVM3 (Myocilin) and W5NQ83 (RNA binding motif protein 25). The ten least abundant proteins in this sample were W5PJG0 (Serum amyloid A protein), W5NX96 (Attractin), W5PV45 (Centrosomal protein of 162 kDa), W5PBY0 (Complement component 4 binding protein alpha), W5P860 (Dual-specificity kinase), P12303 (Transthyretin), W5QFP0 (Thrombospondin 1), W5P3J3 (Complement C1s), W5NXP3 (Serpin A3-6-like) and W5PHP8 (Leucine rich alpha-2-glycoprotein 1).\n\nAll the 236 proteins identified from a composite of all sick sheep serum samples and the 335 proteins from healthy sheep pool (Figure 2B) — taking into consideration overlapping protein IDs, were subjected to gene ontology (GO) classification, within the domain terms of molecular function, biological process, cellular component, protein class using the PANTHER (Protein ANalysis THrough Evolutionary Relationships) classifications system24 (Figure 5). Using the UniProtKB gene identification and mapping tool, 215 of the 236 proteins identified in sick sheep serum were mapped to 206 named gene IDs of sheep. Of these identified sheep genes, only 172 of them were recognised by the PANTHER tool after aligning them to Bos taurus — the species with a genome most closely homologous that of sheep. Likewise, 299 of the 335 proteins identified in healthy sheep serum pool were mapped to 290 sheep genes, however, only 251 genes were recognised in PANTHER based on bovine homologous entries.\n\nNote that the number of genes and individual GO term hits were different between sick and healthy sheep pool.\n\nApart from the number of distinct genes and individual GO term hits, the fractional representation (percentages) of the contributing elements within the domains between sick and healthy sheep serum were comparable (Figure 5). Catalytic activity, binding, structural molecule and receptor activity had comparatively larger representations in the molecular function domain. Cellular process, metabolic process, response to stimulus and localisation had the largest representation in the biological process domain. In the cellular component domain, cell part, extracellular region, organelle and macromolecular complex had the largest representation. As for the protein class domain, enzyme modulator, hydrolase, signalling molecule and receptor terms had the largest representation of the GO terms. It was evident from the molecular function domain that large representations of proteins are involved in catalytic activity (36%) and binding (35%). The biological process domain is interesting for these case studies that involve pathology in that it illustrates inclusion of immune process (5%) and response to stimulus (9%), alongside metabolic (20%) and cellular processes (25%). Cell part (33%), extracellular region (22%) and organelle (20%) GO terms were predominant in the cellular component domain. Meanwhile defense/immunity comprised of only 3%, compared to hydrolase (13%) signalling molecule (12%), receptor (9%) and oxidoreductase (4%) terms in the protein class domain.\n\nProtein pathway analysis was conducted on all the identified proteins from healthy sheep serum sample pool and sick sheep serum using PANTHER (Figure 6). Differences were observed in the enriched protein pathways between sick and healthy sheep. In the results from serum derived from sick sheep, the predominantly represented protein pathways were ATP synthesis, interleukin signalling, angiogenesis, Alzheimer disease-presenilin, integrin signalling, inflammation mediated by chemokine and cytokine signalling, gonadotropin-releasing hormone receptor, cytoskeletal regulation by Rho GTPase, blood coagulation, Huntington disease, p53 pathway, Wnt signalling pathway and Glycolysis pathways. As for the serum derived from healthy sheep, the predominantly represented protein pathways were Alzheimer disease-presenilin pathway, integrin signalling, inflammation mediated by chemokine and cytokine signalling pathway, Huntington disease, Wnt signalling pathway, glycolysis, Parkinson disease, cytoskeletal regulation by Rho GTPase, cadherin signalling, blood coagulation, cholecystokinin receptors (CCKR) signalling map and gonadotropin-releasing hormone receptor pathways.\n\nNote the differences in the enriched protein pathways between samples from sick and healthy sheep.\n\nThe sick sheep serum samples utilised for these experiments were submitted by referring veterinarians for analysis at UQ using automated methods for routine veterinary diagnostic work up. The samples were accompanied by brief presenting clinical case histories presented in Additional file 1.48 Follow-up information on these cases was not available in laboratory entries obtained for this report. With the clinical case history data at hand, the most important of them were signalment (except for two samples (SCi504 and SCi508)) whose ages were missing and the laboratory findings from the analysis of the samples.\n\n\nDiscussion\n\nThe experiments for generating this report utilised SWATH-MS technology to primarily analyse proteins in ex-diagnostic serum samples of sheep suffering from a range of different ailments in comparison with serum from healthy sheep to detect any differences in their protein profiles. The strategy adopted involved testing and validating the potential application of the nascent peptide spectral library built from circulating acellular proteome of sheep.6 The overall intention here was to target protein analytes in significant laboratory findings of the submitted sheep serum samples based on the accompanying clinical data from UQ’s School of Veterinary Science laboratory. The relevant analytes for proteomics studies for this report are found in Additional file 1.48 Among the analytes of interest were gamma-glutamyl transferase (GGT), total protein (TP), serum albumin (Alb), aspartate aminotransferase (AST), alkaline phosphatase (ALP), creatine kinase (CK), fibrinogen and some other analytes that were listed in the reference ranges, for example globulin and serum amyloid. Another important analyte – at least in ruminants – that was not listed in the laboratory panel is haptoglobin.25 These analytes may have appeared as isoforms of their known protein moieties. In some cases however, the genes coding for these proteins were not well defined, possibly due to the consideration that the sheep genome still remains to be fully annotated.26,27 Nevertheless, it is feasible and practical to use draft genome sequences for the interpretation of MS/MS proteomics data without the need for tedious genome annotation.28 This does not negate the point that a well-annotated genome is necessary in order to name genes and proteins unambiguously.29–32\n\nDifferentially expressed serum proteins between healthy and sick sheep were either known or considered to be potential biomarker candidates for diagnosing and monitoring disease states, and this could also provide leads for further research endeavours. There are two important layers to determining the significance of the differences between sick and healthy sheep serum proteomes observed in this work: a) the degree of confidence in the observed difference from an analytical standpoint and, b) the biological plausibility that such differences truly correlate with a disease state. The discussion that follows looks at these preceding two aspects.\n\nA problem of working with the present approach of identifying proteins is that when protein isoforms are not explicitly named and identified, it introduces ambiguity that makes it challenging to identify even well-known proteins as in the instances presented in this report. For example, in the set of samples utilised in this study, Hb was identified as A0A0F6YFJ0 (Beta-C globin). There were also two protein IDs representing GGT which comprised of W5QCX2 (Transglutaminase 1) and W5PB04 (Transglutaminase 3). Serum albumin was identified as W5PWE9 which is yet to be characterised for sheep in UniProtKB. It should be noted that one isoform of albumin, P14639 (Albumin), that was not identified in this set of experiments, has already been characterised in UniProtKB. The ID feature for Aspartate aminotransferase (AST or GOT) was W5PS88. There was no protein ID directly matching ALP in this dataset. However, there were three other phosphatases including W5P195 (Dual specificity phosphatase 14), W5P3B0 (Phosphatidylinositol-3,4,5-trisphosphate 5-phosphatase) and W5QE45 (Serine/threonine-protein phosphatase) that were identified. Meanwhile, CK was represented by two IDs: W5PJ69 (Creatine kinase) and W5NQ67 (Creatine kinase). Fibrinogen was represented by four proteins including W5Q5H8 (Fibrinogen alpha chain), W5NQ46 (Fibrinogen beta chain), W5Q5A6 (Fibrinogen gamma chain) and W5PH03 (Fibrinogen like 1). Serum amyloid was represented by W5PJG0 (Serum amyloid A protein), P42819 (Serum amyloid A protein) and W5PJR0 (Serum amyloid A protein). Haptoglobin was identified as W5P0Q4 (Haptoglobin).\n\nIn veterinary clinical pathology, the determination of the quantity of protein in plasma or serum has always been traditionally based on the amount of albumin and globulin fractions,33,34 and yet there is more to the two broad protein groups when it comes to deep proteomic analysis. For example, in the present set of protein identifications, the globulin fraction was represented by at least 12 protein families or groups. The globulin protein fraction comprised of W5P812 (Protein AMBP), W5PSC1 (Ig-like domain-containing protein), W5Q0R1 (Sex hormone binding globulin), W5NQW4 (Alpha-1-macroglobulin-like), W5PGT9 (Ig mu chain C region), W5QI15 (Ig-like domain-containing protein), W5PSK4 (Ig-like domain-containing protein), A4ZVY9 (Beta-2-microglobulin) W5NSA6 (Alpha-2-macroglobulin), W5PPQ8 (Joining chain of multimeric IgA and IgM), P49920 (Corticosteroid-binding globulin) and the various VH region immunoglobulins such as A2P2G1 and A2P2I1. Being able to identify more proteins in the different fractions of serum proteins gives this approach an edge over traditional clinicopathological protein assays.\n\nThe experiments in this report utilised samples from single sheep case reports as proof-of-concept that SWATH-MS technology can be applied to identify vast numbers of proteins and their alterations in clinical samples of veterinary patients. The comparison of clinical cases (sick sheep serum), versus normalised serum from a large number of healthy sheep is a classical approach typical of biomarker discovery or detection studies.35 With this foundation in place, it is possible to establish a standard for routine proteomic analysis of serum samples submitted to laboratories in future, once a specific baseline serum proteome of sheep has been optimised using far much larger numbers of samples. This method also has the potential to be used for identifying different protein species that show differences between samples. The pooling of samples from healthy sheep was conducted on the premise that the pooled sample would provide a representative proteome of normal sheep serum. Similarly, the rationale of using a pooled sample from sick sheep followed the same premise that that this could provide a representative picture of all the proteins present in the sick sheep, particularly proteins that might only be abundant in specific disease states that could not be detectable in serum samples from healthy individuals. The inherent downside of pooling samples is that this strategy disregards the biological variation of the individuals the samples are drawn from,36 by capturing for example, the ‘average’ proteome profile across a population. And also in the present results, 26 protein IDs were not detected when samples from sick sheep were pooled, as opposed to them being analysed individually (Figure 2B). The reason behind this observation is not immediately clear, but batch effects during sample analysis could be advanced as a contributing factor,37 or possibly unknown effects of pooling samples for downstream analysis. It also follows that the use of pooled serum, at least from normal individuals to act as a control, is an accepted scientific practice for normalising samples that has also been widely used in some human studies.35,36\n\nThe evaluation of chromatographic features of peptides from samples – TICs in this case – was a practical, inexpensive and straightforward visual way of comparing ion intensities between small numbers of samples as previously reported elsewhere.38 Here, only two sample TICs were loaded per displayed panel on PeakView®: one from a sick sheep versus a normalised serum of normal sheep (Additional file 248). The TICs represented a measure of relative abundance of the peptides detected.39 It follows therefore that during interpretation of the TICs, it should be considered that they represented summed signals from the sample as well as background noise.40 The use of TICs in addition to SWATH-MS data extraction has a high depth of analysis whilst factoring in the wide dynamic range in analytes, is a useful strategy especially when considerable differences are expected between samples38 as in the present study. There were distinct differences between TIC profiles of sick sheep serum samples compared to serum profiles from normal sheep; this difference was more marked in the sample from the sheep with scabby mouth lesions (Additional file 2 48). In the majority of the analysed samples, the TIC intensities of sick sheep serum samples were generally higher than those of normal sheep serum, except for the ill-thrift and the ill lamb cases. A possible explanation for this observation could be malnutrition/starvation in the chronically ill sheep, since decreased food intake depletes protein generally and this was even more crucial in the neonatal sheep (ill lamb) that was likely still reliant on colostrum.41–43 Proteins in circulation are either derived from synthesis or from degradation, but considering that the small intestine is the most important organ for synthesis and absorption of proteins in ruminants,44 the detected proteins in starving sheep are therefore most likely to have been derived from tissue degradation. As a result, there was probably a comparatively very little protein reserve to elicit the higher protein intensities compared to the other relatively acute disease case samples. As for the analysis of the pooled sample of sick sheep (SCi511), the TIC profile was generally above that of pooled normal serum up to approximately 38 min when it switched below the pooled normal serum TIC, before peaking again at the hydrophobic end of the chromatogram. An explanation for this observation remains to be established and is open to further interpretation.\n\nThe differences in protein species between individual serum samples from sick sheep could have been due to the different aetiological and pathological factors of the presenting condition in the different sheep that could have stimulated the production of different proteins or their alterations. Without follow-up clinical data on the cases or a definitive diagnosis, it is not possible to fully determine and to explain these differences. Also, clinical cases were from diverse populations which may have contributed to different serum proteomic profiles as proteomes are known to be dynamic.45\n\nDifferent numbers of proteins were identified from the different samples and to some extent, different protein species. The sample from the sheep with scabby foot and mouth lesions had the highest number of protein IDs (Figure 2A). A substantial number of protein IDs were common between sick and the normalised sample from healthy sheep as represented by the ‘pooled healthy control and sick overlap’ legend item in Figure 2A. The protein yield of 210 IDs from the pooled sick sheep sample (SCi511) was much lower than expected as compared to the IDs from all other individual samples considered together. It is not immediately evident as to why this was the case. This relatively low numbers of protein IDs could potentially have been due to sub-optimal tryptic digestion of this particular pooled sample, loss of peptides during sample preparation or probably due to unknown factors that influence the pooling of samples.\n\nThere were differences in protein intensities between the samples as evident in the ten most abundant proteins in each analytical case (Figure 3). The comparison of protein intensities of the pooled serum from healthy sheep (SCi512) versus pooled serum from sick sheep (SCi511) revealed considerable differences for some proteins, with each of the points on the graph representing a protein (Figure 4A). Fewer proteins (210 IDs) were identified in sick sheep sample pool, as compared to 335 IDs in healthy sheep serum pool. It should have also been possible to have a relative quantitation of the proteins centred on their intensities benchmarked on the calibration of the intensities from healthy sheep serum based on the principle of area under curve of the TICs46,47 (Additional file 248). Using this approach, could have demonstrated the feasibility of determining what proteins were either upregulated or downregulated in the clinical serum samples from sick sheep.\n\nThe GO term classification provided an overall picture of the identified proteins in both sick and healthy sheep serum (Figure 5). An important observation from the protein pathway analysis is that it provided 11 clearer protein pathways that peaked in sick sheep serum (Figure 6). The roles of some of these protein pathways in sheep remain to be determined, but they have been studied considerably in humans and other model species such as mice. It is not unreasonable therefore to suggest that with homology, there is a translational potential in the observations made in this study from sick sheep that can be learnt from.\n\nThere were limitations in this experimental study. The first one was that there was only one biological sample for each clinical case study — which is not unusual for case studies. Since each clinical case was unique, to be able to adequately test current observations, three or more technical replicates (or MS injections for that matter) out of each biological sample would have improved the meaningfulness of the data score plot. The second limitation was that it was not possible to re-analyse samples drawn from the same sheep after they recovered (i.e. to act as their own controls), and/or at different stages on the disease to determine if chronicity had an effect on protein species or their alteration – considering that sick sheep samples were opportunistically utilised from laboratory archives at UQ.\n\n\nConclusion\n\nThe use of SWATH-MS analysis successfully identified proteins and enabled protein profiles from serum samples of sick and healthy sheep to be distinguished. It was possible to detect some established clinical biochemical analytes, for example aspartate aminotransferase, creatinine kinase, haemoglobin, serum amyloid A, fibrinogen, haptoglogin, members of gamma-glutamyl transferase group, serum albumin and various proteins of the globulin fraction. Protein pathway analysis provided useful information on the expression profiles of protein groups between sick and healthy sheep by giving them a biological meaning. There was a downside to this approach, for example the detection of alkaline phosphatase – an important analyte that was not obviously identifiable in this dataset. Some detected proteins of sheep such haemoglobin, haptoglobin and some isoforms of serum amyloid did not have distinct gene names as compared to homologous counterparts in related species. Nevertheless, this proof-of-concept study has demonstrated that with a known benchmark or standard samples from healthy individuals, it is feasible to determine relative protein alterations in clinical samples. This approach could have a place in veterinary clinical pathology in future.\n\n\nAuthor’s contributions\n\nThe author originated the concept of the study, conducted the experiments with assistance from staff of The University of Queensland and Queensland University of Technology, and wrote the final manuscript.",
"appendix": "Data availability\n\nPRIDE: Towards a comprehensive targeted proteogenomic assay repository for the liquid fraction of sheep blood. Accession number PXD005002; https://www.ebi.ac.uk/pride/archive/projects/PXD005002.\n\nPRIDE: Application of a novel peptide spectral library using swath analysis for the quantitation of proteins in ex-diagnostic clinical serum samples from sheep. Accession number PXD005077; https://www.ebi.ac.uk/pride/archive/projects/PXD005077.\n\nfigshare: Supplementary data: Interrogation of an ovine serum peptide spectral library to annotate ambiguous clinicopathological biomarkers using data-independent acquisition. https://doi.org/10.6084/m9.figshare.21546999.v8 48\n\nThis project contains the following extended data:\n\n‐ Additional file 1. Clinical data of sick sheep.docx\n\n‐ Additional file 2. Chromatographic features of sheep serum peptide samples.png\n\n‐ Additional file 3. UniProtKB entries of identified proteins.docx\n\n‐ Additional file 4. Relative abundance of proteins in healthy and sick sheep serum.docx\n\n‐ Additional file descriptive notes.txt\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 Australian Red Cross Blood Service for making available the primary samples used in this manuscript under Material Supply Agreement 15-03QLD-19 and staff of The University of Queensland and Queensland University of Technology who provided research degree supervisory oversight for this study.\n\n\nReferences\n\nGillet LC, Navarro P, Tate S, et al.: Targeted data extraction of the MS/MS spectra generated by data-independent acquisition: a new concept for consistent and accurate proteome analysis. Mol. Cell. Proteomics. 2012; 11: O111.016717. PubMed Abstract | Publisher Full Text\n\nAebersold R, Mann M: Mass spectrometry-based proteomics. Nature. 2003; 422: 198–207. 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PubMed Abstract | Publisher Full Text\n\nMi H, Poudel S, Muruganujan A, et al.: PANTHER version 10: expanded protein families and functions, and analysis tools. Nucleic Acids Res. 2016; 44: D336–D342. PubMed Abstract | Publisher Full Text\n\nLepherd ML, Canfield PJ, Hunt GB, et al.: Haematological, biochemical and selected acute phase protein reference intervals for weaned female Merino lambs. Aust. Vet. J. 2009; 87: 5–11. PubMed Abstract | Publisher Full Text\n\nInternational Sheep Genomics CArchibald AL, Cockett NE, et al.: The sheep genome reference sequence: a work in progress. Anim. Genet. 2010; 41: 449–453. PubMed Abstract | Publisher Full Text\n\nJiang Y, Zhang W, Stanton J-A, et al.: The sheep genome illuminates biology of the rumen and lipid metabolism. Science (New York, N.Y.). 2014; 344: 1168–1173. PubMed Abstract | Publisher Full Text\n\nArmengaud J, Trapp J, Pible O, et al.: Non-model organisms, a species endangered by proteogenomics. J. Proteome. 2014; 105: 5–18. PubMed Abstract | Publisher Full Text\n\nGoll J, Montgomery R, Brinkac LM, et al.: The Protein Naming Utility: a rules database for protein nomenclature. Nucleic Acids Res. 2010; 38: D336–D339. PubMed Abstract | Publisher Full Text\n\nBan N, Beckmann R, Cate JH, et al.: A new system for naming ribosomal proteins. Curr. Opin. Struct. Biol. 2014; 24: 165–169. PubMed Abstract | Publisher Full Text\n\nGray KA, Yates B, Seal RL, et al.: Genenames.org: the HGNC resources in 2015. Nucleic Acids Res. 2015; 43: D1079–D1085. Publisher Full Text\n\nFundel K, Zimmer R: Gene and protein nomenclature in public databases. BMC Bioinformatics. 2006; 7: 372. PubMed Abstract | Publisher Full Text\n\nAlberghina D, Giannetto C, Vazzana I, et al.: Reference intervals for total protein concentration, serum protein fractions, and albumin/globulin ratios in clinically healthy dairy cows. J. Vet. Diagn. Investig. 2011; 23: 111–114. PubMed Abstract | Publisher Full Text\n\nAlberghina D, Casella S, Vazzana I, et al.: Analysis of serum proteins in clinically healthy goats (Capra hircus) using agarose gel electrophoresis. Vet. Clin. Pathol. 2010; 39: 317–321. Publisher Full Text\n\nDayon L, Kussmann M: Proteomics of human plasma: A critical comparison of analytical workflows in terms of effort, throughput and outcome. EuPA Open Proteom. 2013; 1: 8–16. Publisher Full Text\n\nBeck HC, Overgaard M, Melholt Rasmussen L: Plasma proteomics to identify biomarkers – application to cardiovascular diseases. Translational Proteomics. 2015; 7: 40–48. Publisher Full Text\n\nČuklina J, Lee CH, Williams EG, et al.: Diagnostics and correction of batch effects in large-scale proteomic studies: a tutorial. Mol. Syst. Biol. 2021; 17: e10240. Publisher Full Text\n\nSchulze WX, Usadel B: Quantitation in mass-spectrometry-based proteomics. Annu. Rev. Plant Biol. 2010; 61: 491–516. Publisher Full Text\n\nDi Girolamo F, Lante I, Muraca M, et al.: The Role of Mass Spectrometry in the “Omics” Era. Curr. Org. Chem. 2013; 17: 2891–2905. PubMed Abstract | Publisher Full Text\n\nCairns DA, Thompson D, Perkins DN, et al.: Proteomic profiling using mass spectrometry--does normalising by total ion current potentially mask some biological differences? Proteomics. 2008; 8: 21–27. PubMed Abstract | Publisher Full Text\n\nYvon M, Levieux D, Valluy MC, et al.: Colostrum protein digestion in newborn lambs. J. Nutr. 1993; 123: 586–596. PubMed Abstract | Publisher Full Text\n\nMellor DJ, Cockburn F: A comparison of energy metabolism in the new-born infant, piglet and lamb. Q. J. Exp. Physiol. 1986; 71: 361–379. PubMed Abstract | Publisher Full Text\n\nMassimini G, Peli A, Boari A, et al.: Evaluation of assay procedures for prediction of passive transfer status in lambs. Am. J. Vet. Res. 2006; 67: 593–598. PubMed Abstract | Publisher Full Text\n\nNeutze SA, Gooden JM, Oddy VH: Measurement of protein turnover in the small intestine of lambs. 1. Development of an experimental model. J. Agric. Sci. 1997; 128: 217–231. Publisher Full Text\n\nPeng J, Gygi SP: Proteomics: the move to mixtures. J. Mass Spectrom. 2001; 36: 1083–1091. PubMed Abstract | Publisher Full Text\n\nKemperman RF, Horvatovich PL, Hoekman B, et al.: Comparative urine analysis by liquid chromatography-mass spectrometry and multivariate statistics: method development, evaluation, and application to proteinuria. J. Proteome Res. 2007; 6: 194–206. PubMed Abstract | Publisher Full Text\n\nMilac TI, Randolph TW, Wang P: Analyzing LC-MS/MS data by spectral count and ion abundance: two case studies. Statistics and its interface. 2012; 5: 75–87. 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}
|
[
{
"id": "238118",
"date": "15 Feb 2024",
"name": "Bart Van Puyvelde",
"expertise": [
"Reviewer Expertise SWATH-MS",
"Viral detection",
"Plasma peptide/protein biomarker screening"
],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 titled \"Interrogation of an ovine serum peptide spectral library to annotate ambiguous clinicopathological biomarkers using data-independent acquisition,\" the author outlines a SWATH data analysis workflow for serum samples from 20 healthy and 7 diseased sheep. While the application of Data-Independent Acquisition (DIA) has been predominantly explored in human specimens, its adoption in veterinary contexts remains limited. I commend the author for the comprehensive detailing of the data analysis workflow; however, the reliance on vendor-commercialized software may constrain its widespread applicability. It is noteworthy to consider the availability of several freely accessible academic pipelines such as DIA-NN and EncyclopeDIA, which have demonstrated superior performance compared to the SWATH MicroApp in terms of identified proteins. Addressing these alternative tools and their potential advantages could enhance the manuscript's relevance. Additionally, given the usage of a pre-existing spectral library, acknowledging its inherent limitations would provide a more comprehensive perspective. A suggestion to expand the impact of the publication involves comparing the performance of various pipelines, including the mentioned academic alternatives, utilizing predicted spectral libraries. This approach has the potential to significantly augment the number of identified peptides and proteins. By incorporating such comparisons, the relevance and impact of the research could be further elevated.\nWhile reviewing the total ion chromatograms of various runs, a significant variance in sample loading caught my attention. Notably, sample B appears to have a sample load approximately 10 times higher than the other samples. Without any batch correction, such as normalization, performing a meaningful differential analysis becomes challenging. Moreover, the inclusion of only seven serum samples from individuals with different underlying diseases, as emphasized by the author, raises concerns about the study's robustness. In my view, a more controlled and expansive disease cohort is essential to draw conclusive insights regarding the annotation of clinicopathological biomarkers from this study. If the authors intend to include such a claim in the title, it would necessitate substantial additional work, which may often be impractical. Therefore, I recommend modifying the paper's title to accurately reflect the study's scope and limitations.\nSome of the figures (Figure 2A, 5 and 6) can also be moved to supplementary as they don't really add a lot of information.\nMinor comments: -Page 5: cold acetone (20°C) => -20°C -Page 5: is centrifugation at 4,000g for 2 minutes sufficient to precipitate the proteins? -Page 5: Aceto nitrile => acetonitrile -Page 5: What kind of nanoLC instrument was used? Provide some more details -Page 5: 36 windowsl => remove the -l -Page 5: instrument as described in earlier => remove the \"in\" - Page 14 :Why did the author decide to pool the samples prior to trypsin digestion instead of pooling them after digestion?\"\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Partly\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nNot applicable\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes",
"responses": []
},
{
"id": "243204",
"date": "01 Mar 2024",
"name": "Uma Aryal",
"expertise": [
"Reviewer Expertise Quantitative proteomics",
"phosphoproteomic",
"application of proteomics to senescence or aging 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\nData Independent Acquisition (DIA) is gaining traction in recent years and more and more laboratories are adopting these methods instead of the most widely used DDA method. Thus, analysis of ovine serum peptides spectral library is timely manuscript. Overall, the manuscript is written well and easy to follow. However, I have several comments or concerns, which I hope author will find useful and addressing them will improve the quality of the manuscript. First, I found number of proteins identified in this study significantly low compared to other similar studies in other animals. The study identified only 335 and 236 proteins in healthy and sick serum samples, which seems significantly low. In our own experience using DDA, we can easily profile close to 1000 proteins in mouse or dog plasma or serum samples. Some clarification in the low number of protein IDs would be helpful. My understanding is that DIA will give more IDs compared to DDA method.\n\nMy second concern is the healthy and sick samples. It appears that healthy samples were obtained commercially while sick samples were obtained clinically that presented a range of clinical situations. I am not sure comparing commercial samples with the clinically collected samples is a good way to profile protein differences. This may present significant heterogeneity. Based on my own experience, animals raised in different environment and fed different food exhibit significant differences in serum proteome profile. It would be nice to describe these limitations briefly in a separate paragraph. It was not clear to me how protein families grouped together. Was this done based on unique and/or shared peptides sequences? Also, the term \"unique proteins\" may be misleading. It would be better to refer them as \"proteins identified only in xxx, etc. Also, low overlap of proteins between healthy and sick sheep also caught my attention. I assume some of these differences could have been originated from samples coming from totally different environment and in addition to disease states, other factors might have contributed these discrepancies. Again, a separate section summarizing all these limitations will be good service to the readers. Details of downstream data analysis and statistical analysis to identify differentially abundant proteins was not provided. I suggest the author to provide this information in detail.\n\nIs the work 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/11-1433
|
https://f1000research.com/articles/11-501/v1
|
05 May 22
|
{
"type": "Case Study",
"title": "Implementation and assessment of an end-to-end Open Science & Data Collaborations program",
"authors": [
"Huajin Wang",
"Melanie Gainey",
"Patrick Campbell",
"Sarah Young",
"Katie Behrman",
"Melanie Gainey",
"Patrick Campbell",
"Sarah Young",
"Katie Behrman"
],
"abstract": "As research becomes more interdisciplinary, fast-paced, data-intensive, and collaborative, there is an increasing need to share data and other research products in accordance with Open Science principles. In response to this need, we created an Open Science & Data Collaborations (OSDC) program at the Carnegie Mellon University Libraries that provides Open Science tools, training, collaboration opportunities, and community-building events to support Open Research and Open Science adoption. This program presents a unique end-to-end model for Open Science programs because it extends open science support beyond open repositories and open access publishing to the entire research lifecycle. We developed a logic model and a preliminary assessment metrics framework to evaluate the impact of the program activities based on existing data collected through event and workshop registrations and platform usage. The combination of these evaluation instruments has provided initial insight into our service productivity and impact. It will further help to answer more in-depth questions regarding the program impact, launch targeted surveys, and identify priority service areas and interesting Open Science projects.",
"keywords": [
"Open Science",
"Metascience",
"Academic Libraries",
"Program Assessment",
"User Data"
],
"content": "Introduction\n\nThe ways in which research is conducted are shifting toward more open, transparent and collaborative practices (McKiernan et al., 2016). This trend has been a response to changes in the funding and publishing landscape, the nature of research collaboration, the emergence of digital research infrastructures and cultural shifts in scientific practice. The term ‘Open Science’ has been used as an umbrella term to describe these trends. In 2018, Vicente-Saez and Martinez-Fuentes arrived at the following formal definition of Open Science through an analysis of ten years of scholarly literature on the topic: “[T] ransparent and accessible knowledge that is shared and developed through collaborative networks” (Vicente-Saez and Martinez-Fuentes, 2018, p. 434). Similarly, Fecher and Friesike proposed five schools of thought that capture the breadth and complexity of the Open Science discourse, namely, schools focused on infrastructure, collaboration, public access to research, impact measurement and democratic principles (Fecher and Friesike, 2014, p. 20).\n\nThe shift towards more openness in research depends on many factors, including cultural and behavioral shifts amongst researchers, changes to incentive structures and publishing models, and infrastructural developments. While many research communities recognize the value of Open Science for furthering scientific knowledge, in actual practice, openness in research has been much more challenging to achieve (Nosek et al., 2015). Funders, publishers, and the public all play key roles in moving research toward open practices, as do institutions of higher education where incentive structures may run counter to a culture of research transparency. Despite this, there are various stakeholders in higher education settings that can foster Open Science practices. Moreover, Open Science overlaps with different areas of support across a university. For example, entities dealing with research integrity may take on the promotion of Open Science through a research transparency lens (Bouter, 2018). Institutional research and analysis offices may have an interest in Open Science practices, as Open Science tools and platforms can assist with measuring and tracking research impact (De Castro, 2018). Open Science initiatives may sprout from disciplinary or cross-disciplinary projects or sit within computer or data science departments. Examples of such initiatives include Stanford’s multi-school Center for Open and REproducible Science (CORES) and the Berkeley Initiative for Transparency in the Social Sciences (BITTS).\n\nAs Open Science has matured, academic libraries have also entered this space leveraging the natural alignment with existing services and principles related to information access and dissemination. For example, many libraries provide both infrastructure (e.g., institutional repositories) and funding (e.g., open access publishing funds) for sharing the products of research. Libraries also commonly provide training and support for managing research data, which relates to Open Science through the facilitation of practices that support data sharing and reuse. Recent literature suggests that libraries recognize their role in the Open Science movement, particularly in relation to repositories and open access publishing (Ogungbeni et al., 2018). Ayris and Ignat discussed important roles for libraries in Open Science in Europe (Ayris and Ignat, 2018), and other research indicates a growing role for libraries in the Open Science landscape in Africa (Siyao et al., 2017; Tapfuma and Hoskins, 2019). Nonetheless, to our knowledge, the formalization of these tools and services in the form of “Open Science programs” in academic libraries is rare. Moreover, most libraries are likely not yet building programs with goals of providing a suite of tools and services to support Open Science throughout the research lifecycle.\n\nHere, we present the framework for a novel Open Science program established in 2018 at Carnegie Mellon University (CMU) Libraries. The program, called Open Science and Data Collaborations (OSDC), encompasses a range of activities, tool support and training addressing Open Science practices throughout the research lifecycle (Figure 1 and Table 1). Like other libraries, CMU Libraries also provide an institutional repository, a fund to partially cover author processing changes for open access publishing, and research data management services. While these services operate outside of the OSDC program umbrella, the programs and services work hand-in-hand to facilitate end-to-end Open Science practice. The purpose of the current work is to present this model for a library-based Open Science program with a focus on program metrics and assessment. We begin with a brief environmental scan of Open Science activities at peer institutions. We follow with a logic model outlining our program activities, as well as short-, mid-, and long-term goals, and present examples of metrics that can be used and gathered to measure success. We conclude with a brief discussion of future implications for program planning and evaluation.\n\nTools and services that OSDC supports with consultations, training opportunities, or licenses are mapped onto the phases of the research life cycle. Services and tools in gray boxes are supported by colleagues in the University Libraries that specialize in Open Access, Research Data Management, and Open Educational Resources. OER: Open Educational Resources; APC: Article Processing Charge; KiltHub: CMU’s institutional repository; AIDR: Artificial Intelligence for Data Discovery and Reuse Conference; dataCoLAB: Data Collaborations Lab, an initiative to foster partnerships on data science projects on real-world research data.\n\nServices in the OSDC program are composed of four major categories: tools, training, events, and collaboration.\n\n\nEnvironmental scan\n\nTo evaluate the landscape of library Open Science programs, in the spring of 2021 we conducted an environmental scan of Carnegie Mellon University’s peer institutions, a list of 13 institutions of common qualities and goals (as defined by the Office of Institutional Research and Analysis at the university) (Table 2). From the websites of each individual institution and each institution’s library, we searched for the general terms “open science,” “open scholarship,” and “open research” to attempt to locate similar programming and services to those offered by OSDC at CMU. We also searched for traditional Open Access resources, such as an institutional repository and an institutional Open Access policy to benchmark the number of peers with general Open Research services that may not be specifically described as “Open Science.” While the majority of peer institutions support open scholarship through open access policies and institutional and data repositories, dedicated open science centers and programming, either through the university library or through departmental structures, are less common (Table 2).\n\nCMU’s peer institutions are California Institute of Technology, Cornell University, Duke University, Emory University, Georgia Institute of Technology, Massachusetts Institute of Technology, Northwestern University, Princeton University, Rensselaer Polytechnic Institute, Rice University, Stanford University, University of Pennsylvania, Washington University in St. Louis.\n\nIn addition to manually checking the websites of peer institutions and to identifying any related programs outside of peer institutions, we ran a Google search using the following search string, which queried sets of search terms within three words of other search terms and limited the results to websites of U.S.-based postsecondary institutions: “open|reproducible|reproducibility AROUND(3) research|science|scholarship AROUND(3) institute|center|program” site:.edu. We then reviewed the results of the search until no relevant results were found on five consecutive results pages. No additional dedicated Open Science programs were identified with the Google search.\n\n\nProgram implementation\n\nIn 2017, we began to develop services and initiatives to support open and reproducible research in response to the growing need for reliable infrastructure and training for Open Research practices (Mckiernan et al., 2016; Nosek et al., 2015; AAU-APLU Public Access Working Group Report and Recommendations, 2017). What began as an ad hoc collection of services and collaborations was formalized as the Open Science & Data Collaborations (OSDC) Program in 2018. This program within Carnegie Mellon University Libraries consists of a team of subject librarians with deep research expertise and specialists in research data management and Open Data. While we have adopted the name “Open Science” due to its common use in the community, we support all types of research and often use the term “Open Research” to describe our activities. The OSDC program provides training and support for tools and practices that can be mapped onto the phases of the research life cycle (Figure 1). Since our services together cover the entire life cycle, we describe the program as providing “end-to-end” support. The program has been in a phase of rapid expansion since its inception in 2018. We have leveraged our research experience, particularly in the life sciences, and our existing campus partnerships to develop new services that we believe will be of use and interest to the CMU community and help make research products open in accordance with the FAIR principles (Wilkinson et al., 2016).\n\nPrior to the development of the OSDC program, CMU Libraries already provided extensive support for some areas of scholarship that are typically defined as Open Science, such as Open Access publishing (Fecher and Friesike, 2014). Our comprehensive institutional repository, KiltHub, also predates the creation of OSDC. Currently, we collaborate with colleagues in the library that specialize in open access, research data management (RDM), and open educational resources (OER) to provide holistic support for open scholarship. These areas of Open Science that are outside of the purview of the OSDC program are not currently assessed by us (Figure 1). In spite of the fact that KiltHub existed prior to the development of OSDC, we currently help support the platform and assess its usage as an integral piece of infrastructure for data sharing.\n\n\nProgram assessment\n\nAs OSDC expands, one challenge has been getting structured and actionable feedback from the CMU research community, particularly from disciplines outside of the life and social sciences. To this end, we created a new arm of the program in 2021 that focuses on research and assessment. Our recent work has focused on developing a logic model and quantitative metrics on tool usage and event and training attendance. We will use this multi-pronged assessment approach to identify gaps in our service, shape the growth of the program in a data-driven and user-centered manner, identify future members for our Advisory Board, and create surveys designed for specific segments of our user community. Keeping the user in mind will be critical as the needs of the research community continue to evolve against the dynamic backdrop of data sharing mandates and the increasing desire for transparency and reproducibility in the research community.\n\nThe first component of our assessment strategy is a logic model (Newcomer et al., 2015) that provides a snapshot of the activities offered by the OSDC program and their respective outputs, resources needed to run the program, short-, medium-, and long-term goals to achieve for our users, and a list of partnerships formed through the program (Figure 2). It provides a bird’s-eye view of the activities of the program and guides operational decisions and strategic planning. Values in outputs are estimated and serve as a baseline for further assessment. It should be noted however that values are not comparable between tools due to different time frames used for component datasets. The logic model will be reevaluated yearly.\n\nA logic model was created by listing inputs, activities, outputs, outcomes, and partners for each activity and creating a narrative. A simplified graphic summary was created to represent essential elements of the logic model. Inputs: resources required for all activities. Activities: the five groups of activities in the OSDC program; from top to bottom: tools, workshop, events, collaboration, and outreach. Outputs: product of each activity. Outcomes: short-, medium-, and long-term goals. Partners: partnerships formed to date.\n\nTo find more quantitative ways to measure program impact, we developed the second component of our current assessment strategy, a “5W1H” (Who, What, When, Where, Why, How) framework. Using this framework, originally developed for communication action research (Yoshioka et al., 2001), we developed metrics that use tool usage and event attendance data to help answer questions about our users and their use of our services.\n\nFirst, we collected existing usage data across tool platforms. Specifically, we gathered usage data for the following tools: KiltHub, Open Science Framework, protocols.io, LabArchives. We also collected event registration data for Open Science-themed Libraries workshops, Carpentries workshops, Open Science Symposium, AIDR (Artificial Intelligence for Data Discovery and Reuse) Conference, and dataCoLAB (Data Collaborations Lab). We used event registration data as a proxy for event attendance since attendance data were not consistently collected. We expect, however, that registrations for events are higher than the actual attendance. Finally, engagement with the Open Science Newsletter, one of our core marketing tools, was also included in the assessment. The details of how data were collected for each of these services can be found in the Data Collection Methods section of this paper.\n\nData across platforms and events were cleaned and aggregated into a master dataset. The protocol we used to create the master dataset is published on protocols.io. We used Andrew IDs (CMU institutional emails) as unique identifiers for users of our services. Since the KiltHub dataset includes institutional and departmental affiliation data for all current CMU graduate students, staff, and faculty, we matched Andrew IDs for users of our other services to the KiltHub dataset. If users provided non-institutional email addresses, we queried their names in the CMU directory to determine their Andrew IDs, if possible. Undergraduates are represented in the dataset simply as “Undergraduates” since we could not consistently determine their departmental affiliation. We confirmed their status as undergraduates by querying their names in the CMU directory.\n\nCMU and University of Pittsburgh (Pitt) have some joint centers and programs, and we noted that 60 users in the master dataset (5% of the total 1,348 unique users) have primary Pitt affiliations (Table 3). For our analyses, we filtered out Pitt users. We also filtered out users from other non-CMU institutions or unidentified institutions (33%) and CMU users if we could not determine their departmental affiliation or if they were affiliated with administrative units on campus (4%). The total remaining records represented in the Results section (n=787) represents 58% of the total unique records (n=1,348) with which we began.\n\nIn our analyses, we only included Carnegie Mellon University (CMU) users with known departamental affiliations (n=787). CMU users with unidentified or administrative affiliations (n=56), University of Pittsburgh users (n=60), or users at other or unidentified institutions were filtered out of the dataset (n=445).\n\nBased on the master dataset and platform-specific data, we generated a list of meaningful questions within the 5W1H framework (Table 4). Metrics and their sub-variables were then defined to answer those questions. Currently, we are focusing on questions that we are able to answer readily with the data at hand, e.g.: who uses our tools and participates in our activities, which disciplines are the most engaged, and how do people use our tools and activities? Most of the metrics related to the questions require data collected from platform dashboards or provided by vendors. In other cases, the metric was derived from the dashboard or vendor data with simple calculations. For example, we can use data from the KiltHub dashboard to determine the institutional and departmental affiliation of each user. We can then derive the stage of the career of the user by querying institutional email addresses in the CMU directory. It should be noted that while we know that our users are largely in the Pittsburgh area, we do not collect any other information, such as IP addresses, that could be used to answer “Where” questions. Additional data collection is required to answer more nuanced questions about the impact and value of our services for users. Questions we eventually hope to address include: why do people use our tools or activities, how much value did we provide to users, and what impact are we making in people’s research process and in the whole research ecosystem?\n\nMetrics being used to evaluate the performance of the Open Science and Data Collaborations (OSDC) program and the variable(s) that are used to calculate each one. Metrics are organized using a “5W1H” (Who, What, When, Where, Why, How) framework representing the major classes of query the dataset is designed to answer. Data for each metric can either be collected directly from dashboards, vendors, or registration records, or derived from the direct data with simple calculations.\n\n* Metric that we have partial data for and can be calculated in the future.\n\nImportantly, the metrics can be applied to the program as a whole or to specific tools and services. The user affiliation metric will indicate whether we are achieving broad coverage of disciplines with the program. The superuser metric will help us identify Open Science advocates in our campus community that can support our outreach efforts and provide valuable feedback. We can also track adoption of specific services over time with our Growth Rate metric to examine trends in Open Science research and help guide decision making.\n\n\nExample applications of current metrics\n\nEven though the framework is still a work in progress, limited by the state of existing data, it already allows us to ask simple questions. As a proof of concept, we provide a few examples of applying this framework to extract interesting patterns from existing data.\n\nTo obtain an overview of disciplinary engagement, we summarized the number of users for each department, based on their primary affiliations (Figure 3). The data came from the integrated dataset where usage of a given service or activity was represented as a “true/false” value. A user with a “true” in any of the services as counted as 1. These data show that the Heinz College of Information Systems and Public Policy has the highest number of users, followed by the Biological Sciences Department, University Libraries, and the Psychology department. We think this result can be partially explained by disciplinary culture as these disciplines are traditionally more engaged with library services and more active in the Open Science movement. Interestingly, some engineering and computer science departments also have high numbers of users, suggesting that we are starting to generate buy-in from these disciplines.\n\nNumber of users by department or academic unit, based on their primary affiliations. The main dataset integrating all usage data was used as input. Each user is counted only once even if they use multiple services. CNBC: Center for the Neural Basis of Cognition, CEE: Department of Civil and Environmental Engineering, ECE: Department of Electrical and Computer Engineering, MSE: Department of Materials Science and Engineering, Dietrich: Dietrich College of Humanities and Social Sciences, Heinz: Heinz College of Information Systems and Public Policy, HCII: Human Computer Interaction Institute, INI: Information Networking Institute, ICES: Institute for Complex Engineered Systems, IPS: Institute for Politics and Strategy, ISRI: Institute for Software Research, iii: Integrated Innovation Institute, LTI: Language Technologies Institute, MBIC: Molecular Biosensor and Imaging Center, PSC: Pittsburgh Supercomputing Center, SDS: Social and Decision Sciences, SEI: Software Engineering Institute, Tepper: Tepper School of Business.\n\nThe number of users of each department does not necessarily reflect how active users from these departments are. Using KiltHub as an example, we further dissected the level of user activity for each individual tool hosted by the program. The reason we did not use the integrated master dataset for this purpose is that measurements between platforms, e.g., number of notebooks or number of registrations, are not comparable with each other. A breakdown of the number of users on KiltHub revealed that Software Engineering Institute (SEI), Psychology, and University Libraries, again, were among the departments or academic units that have the greatest number of KiltHub users (Figure 4A, blue bars). However, when looking at user activity levels, specifically public items owned by users, those from SEI collectively owned fewer items compared to those from Psychology and University Libraries (Figure 4A, red line). We further analyzed KiltHub usage at the level of individual users and saw different departments emerge when compared to the result from the total number of users. Among the top 10 departments ranked by the median values of the number of public items owned by each user in a given department, the School of Business ranked the highest, followed by University Libraries and the Computer Science Department (Figure 4C). Even though the median values were relatively low overall – less than five items per user – some users owned much higher numbers of public items on KiltHub (Figure 4C, outliers). This trend was also reflected at the level of the individual user, with the most active users owning more than 20 public items on KiltHub while the majority of users owned less than five items (Figure 4B). We define users with more than 10 public items as “superusers.” We were able to identify these users (anonymized in this manuscript) and their department affiliations (Figure 4D). The ability to identify superusers is especially valuable for collecting targeted feedback with interviews and surveys for service improvements in the future.\n\n(A) Departmental breakdown of number of KiltHub users (blue bars) and number of public items owned by users collectively in these departments (red line). (B) Frequency of number of public items owned per user (frequency is measured by the count of users with the specified number of public items owned). (C) Boxplot showing the distribution of the number of public items owned per user for the top 10 departments, measured by mean number of items per user. The boxed area represents the interquartile range (IQR), with the lower bar representing the first quartile (Q1) value, the intermediate bar representing the median value (Q2), and the top bar representing the third quartile (Q3) value. The lines, or “whiskers”, extending above and below the boxed area represent the range of values contained within 1.5 times the interquartile range (1.5 x IQR). Points extending beyond the whiskers represent outlier values (> 1.5 x IQR). (D) Number of public items owned for the 10 most active users (items > 10). These users are identified by their User ID (autonumber value assigned by Excel) to conceal the users’ identities. CNBC: Center for the Neural Basis of Cognition, CEE: Department of Civil and Environmental Engineering, ECE: Department of Electrical and Computer Engineering, MSE: Department of Materials Science and Engineering, Heinz: Heinz College of Information Systems and Public Policy, HCII: Human Computer Interaction Institute, INI: Information Networking Institute, ISRI: Institute for Software Research, LTI: Language Technologies Institute, SEI: Software Engineering Institute, Tepper: Tepper School of Business.\n\nAn important indicator of a program’s success is its growth over time. We used tool usage over time as a proxy to explore this question. By examining the total number of users who deposited data on KiltHub or the total number of accounts on LabArchives and protocols.io over time (Figure 5), we found that there has been a steady increase in use every year since the inception of the OSDC program. This initial analysis establishes a useful baseline for future longitudinal studies.\n\nNumber of total depositors on KiltHub, user accounts on LabArchives, and user accounts on protocols.io increased each year since the beginning of the program in 2018.\n\n\nDiscussion and future directions\n\nData sharing has represented a massive paradigm shift for research in recent years (Gewin, 2016). There are varying disciplinary norms and attitudes around data sharing and researchers often lack the training, time, infrastructure, or perceived incentives to openly share their research products. Fear that the data will be misused is another common concern (Fecher et al., 2015; Tedersoo et al., 2021; Tenopir et al., 2015). To address these challenges, we have created one of the first end-to-end Open Science programs sponsored by a library, with services that map onto all phases of the research lifecycle. One of our guiding priorities for creating Open Science services is that they have an impact on fostering collaboration and a cultural change towards research transparency. It is important, however, that we remain mindful of the barriers that researchers face. We therefore support a full gradient of Open Science practices, ranging from sharing research products publicly to improving the reproducibility of private workflows. For example, for researchers that are unable to share data due to working with sensitive data types, or are simply uncomfortable with data sharing, we might improve the reproducibility of their workflow for their future selves and collaborators. These types of consultations provide us with valuable opportunities to not only improve researcher experience around Open Science, but also discuss the benefits of publicly sharing research. Together with our community-building events, these types of interactions with researchers allow us to foster a culture of transparency.\n\nAs we continue to create services, we need to rely not only on conversations with researchers, but also on periodic quantitative assessments to understand their impact. The work presented here is the beginning of our program assessment and provides methods that we will update periodically and eventually supplement with additional metrics. This will allow us to focus our resources on priority areas, maximize the efforts of our small team, and guide our efforts to secure funding.\n\nMost of the data currently in our possession focuses on event registration and tool usage. Registration data is useful primarily for providing insights into, e.g., the popularity of specific OSDC initiatives (number of registrants, frequency of use, etc.), the reach/coverage across CMU and broader research community, specifically with regard to user type (student, faculty, etc.), institutional and departmental affiliation, and potential superusers. Our current data also include several variables related to the effectiveness of our various initiatives, e.g., number of items on KiltHub, number of projects and registrations on OSF, number of notebooks or activities on LabArchives, event attendance, or open and click rate of the Newsletter. However, we are only scratching the surface about the effectiveness or impact of the various OSDC initiatives; many deeper questions, e.g., how many publications, grant applications, career opportunities that we help users to obtain, and how much time we save users in their daily research, cannot be answered with the existing metrics. Importantly, the lack of these sorts of metrics prevents us from being able to make a clear value proposition to researchers for whom productivity, efficiency, and impact are the most important factors.\n\nDespite these limitations, the current data and the 5W1H metrics framework will serve as a baseline to develop a strategy for data management in the future to guide data collection, update, and analysis. A large part of our data collection process is limited by the platforms or tools that host the data. However, the usefulness of data can be improved by a few tweaks. To get the most out of our usage and registration data, a date field should be included for all relevant data tables, which will allow us to infer, for example, how the number of link clicks in a particular issue of our newsletter influences the number of registrations for specific events. More generally, date information can reveal temporal patterns in the use of various tools/platforms and attendance at particular events, allowing us to better target our outreach efforts and workshops. Adding a date field will also allow us to track more meaningful changes in use after controlling for natural fluctuation patterns, which can in turn be used to estimate programmatic growth or decline.\n\nTo develop a more mature data management system, metrics should be developed to provide insight into different stages of the research lifecycle (Figure 1), particularly around the issues of productivity, efficiency, and impact. The specific variables that are relevant in each case will depend on the particular stage of the research lifecycle we are considering. For example, usage of protocols.io would likely reflect the data collection and analysis stage, while KiltHub usage more likely reflects the publishing and sharing stage.\n\nWe would also like to develop a more systematic data collection strategy that allows regular updates to data and results. The current data collection, cleaning, and analysis process is highly manual, making it time-intensive, error prone, and difficult to update. Developing an automated or semi-automated workflow would help to ease the administrative overhead on data updates and enable us to ask more longitudinal questions.\n\nThe combination of the logic model and the 5W1H framework provides complementary instruments to evaluate our program’s impact and to inform decision making. The logic model provides a bird’s-eye view of program activities and is an ideal tool for goal setting and communicating higher level ideas with leadership and stakeholders. The 5W1H framework, on the other hand, helps to evaluate and understand our activities and user engagement at a more granular level, making it possible to quantitatively assess our successes, identify areas for improvement, prioritize future work, and refine outreach strategies.\n\nThe most difficult thing in the metrics framework is the “why” question: what are the motivations for people to use our services? Is it to meet funder/publisher mandates, to get credit, or for other reasons? Developing such metrics would make it possible to quantitatively assess user motivation and productivity, evaluate the value and success of our services, and identify areas for improvement and prioritization in the future. For these types of questions, we would like to get direct feedback from users through surveys and interviews. To this end, the “superuser” metric (Figure 4D) in the 5W1H framework helps to identify the right users to reach out to. We had initial success applying this metric to form a OSDC Advisory Board from our users, composed of graduate students, postdoctoral fellows, and faculty who are Open Science advocates and practitioners, and represent a variety of disciplines. The group meets 3-4 times a year to provide feedback in the style of a focus group on service updates, outreach strategies, and disciplinary practices and challenges.\n\nOur work on the implementation of an end-to-end Open Science program and the development of assessment instruments will serve as a model that can be adopted by Open Science programs at other institutions, or other service-oriented organizations that wish to evaluate their success and impact. With further enrichment and adoption, the combined logic model and 5W1H framework we developed has the potential to grow into a benchmarking tool for equivalent programs and products that require both qualitative and quantitative assessment.\n\n\nData collection methods\n\nKiltHub. For the master dataset, profiles of all active users on or before 2 April 2021 were downloaded from the KiltHub Admin dashboard. We used the following data fields from the profiles for this study: ID, first and last name, email address, affiliation (department or center), and number of public items owned. Only data depositors with more than one public item owned were included in data analysis, while the names and email addresses of all users were used for data harmonization (see the published protocol for details). We filtered out private items since there are many reasons why a user might choose to keep their projects private. For usage over time, a separate dataset was downloaded from the dashboard that contains information about depositors.\n\nprotocols.io. Usage data including number of users, private, protocols, and public protocols were provided quarterly by the vendor and were collected for this study on 30 November 2021. Per protocols.io privacy policies, identifying information such as names, email addresses, or departmental affiliations were not shared. Therefore, these data were not included in the User Summary in Figure 3.\n\nOpen Science Framework (OSF). We collected user data from Open Science Framework (OSF) with our institutional OSF dashboard that includes first and last names and number of public projects and registrations on 19 January 2021. The number of public projects and registrations per user is the sum of these two metrics. Institutional emails were gathered by querying names in the Carnegie Mellon University Directory.\n\nLabArchives. A Detailed Usage Report was downloaded from the Site Administrator dashboard. The report included first and last names, institutional email address, type of account (CE type), number of notebooks, and number of activities. For the purpose of this study, we were interested in Researcher and Instructor accounts. Student accounts were filtered out of the dataset. Data for the User Summary in Figure 3 were collected on 20 January 2021 and the usage over time data in Figure 4 were collected on 20 November 2021.\n\nNewsletter. Newsletter data was accessed through Mailchimp. We were interested in users that routinely open the newsletter. To gather these data, we navigated to the Audience Dashboard and selected the Often segment under Engagement. This allowed us to collect data on our most engaged users, including first and last names and email addresses. We then searched user profiles in Mailchimp to gather data on Open Rate and Click Rate for each user. Newsletter data were collected on 20 January 2021.\n\nEvents. Event registration data for Open Science Symposium and AIDR were collected from the Indico, EasyChair, and EventBrite platforms. The data collected for each registrant included event name, first and last names, email address, and institution. Participation data from dataCoLAB were collected using a project intake form in Google Forms.\n\nWorkshops and training. Workshops and training related to Open Science at CMU are delivered primarily through two formats: (1) one- to two-hour workshops offered through the Libraries’ workshop series on the following topics: OpenRefine, Jupyter Notebooks, Open Science Framework, Data Management, and R, and (2) Carpentries workshops, which are two-to-three-day training sessions organized and managed by the Libraries’ Carpentries organizing team. Registration data were collected for Libraries’ and Carpentries workshops from LibCal and Eventbrite, respectively. Registration data, including first and last names and email addresses, were collected for each occurrence of a workshop that had occurred by 1 January 2021. All Libraries’ and Carpentries workshop data were combined for each workshop type (type defined by a combination of format and topic). Users were merged if they had used different emails for registration for different workshops, and it was clear from their name that they were the same person. For users that had registered for multiple occurrences of the same Libraries’ workshop, it was assumed that they had only attended one. For Carpentries workshops, we assumed that registrants may have attended more than one workshop even if it covered the same content. Libraries’ workshop data were then combined into a single dataset indicating whether or not a user had registered for a given workshop type and the total number of workshop types attended by each user.\n\nAfter extensive communication with the Institutional Review Board (IRB), it was advised that as this project is intended for evaluation and improvement of internal processes without making generalizing statements, did not fall under the definition of research, and therefore did not require IRB approval. Informed consent for collecting the original data hosted by the university and the libraries was obtained by the university’s legal office. Data have been anonymized for this study before collection and analysis. Anonymizing the data does not change the scientific meaning of our findings.\n\n\nData availability\n\nBecause original data used to develop assessment methods contain identifiable user information, they are only for internal use. Deidentified and aggregated data are openly available in KiltHub, CMU’s institutional repository (DOI: https://doi.org/10.1184/R1/19438586). Protocols used for data cleaning and processing are openly available on protocols.io (https://doi.org/10.17504/protocols.io.b29gqh3w).",
"appendix": "Acknowledgments\n\nWe thank Hannah Gunderman and Emma Slayton for contributing workshop data. We thank Brian Mathews for helping to develop the logic model.\n\n\nReferences\n\nAssociation of American Universities (AAU) and Association of Public and Land-grant Universities (APLU): AAU-APLU Public Access Working Group Report and Recommendations.2017. Reference Source\n\nAyris P, Ignat T: Defining the role of libraries in the Open Science landscape: A reflection on current European practice. Open Inf. Sci. 2018; 2(1): 1–22. Reference Source\n\nBouter LM: Fostering responsible research practices is a shared responsibility of multiple stakeholders. J. Clin. Epidemiol. 2018; 96: 143–146. PubMed Abstract | Publisher Full Text Reference Source\n\nCampbell P, Wang W, Gainey M, et al.: Cleaning, Aggregating, and Filtering CMU Libraries Open Science and Data Collaborations Program Data. protocols.io. 2022. Publisher Full Text\n\nDe Castro P: The Role of Current Research Information Systems (CRIS) in Supporting Open Science Implementation: The Case of Strathclyde. Informačné Technológie a Knižnice, Special Issue 2018. 2018; 21–30. Publisher Full Text\n\nFecher B, Friesike S: Open science: One term, five schools of thought. Open. Sci. 2014: 17–47. Publisher Full Text Reference Source\n\nFecher B, Friesike S, Hebing M: What Drives Academic Data Sharing?. PloS One. 2015; 10(2): e0118053. PubMed Abstract | Publisher Full Text\n\nGewin V: Data sharing: An open mind on open data. Nature. 2016; 529(7584): 117–119. Publisher Full Text\n\nMckiernan EC, Bourne PE, Brown CT, et al.: How open science helps researchers succeed. elife. 2016; 5(JULY). PubMed Abstract | Publisher Full Text\n\nNewcomer KE, Hatry HP, Wholey JS: Handbook of practical program evaluation. 2015. Reference Source\n\nNosek BA, Alter G, Banks GC, et al.: Promoting an open research culture. Science (New York, N.Y.). 2015; 348(6242): 1422–1425. PubMed Abstract | Publisher Full Text Reference Source\n\nOgungbeni JI, Obiamalu AR, Ssemambo S, et al.: The roles of academic libraries in propagating open science: A qualitative literature review. Inf. Dev. 2018; 34(2): 113–121. Publisher Full Text\n\nSiyao PO, Whong FM, Martin-Yeboah E, et al.: Academic libraries in four Sub-Saharan Africa countries and their role in propagating open science. IFLA J. 2017; 43(3): 242–255. Publisher Full Text Reference Source\n\nTapfuma MM, Hoskins RG: Open science disrupting the status quo in academic libraries: A perspective of Zimbabwe. J. Acad. Librariansh. 2019; 45(4): 406–412. Publisher Full Text Reference Source\n\nTedersoo L, Küngas R, Oras E, et al.: Data sharing practices and data availability upon request differ across scientific disciplines. Sci. Data. 2021; 8(1): 192. PubMed Abstract | Publisher Full Text\n\nTenopir C, Dalton ED, Allard S, et al.: Changes in Data Sharing and Data Reuse Practices and Perceptions among Scientists Worldwide. PloS One. 2015; 10(8): e0134826. PubMed Abstract | Publisher Full Text\n\nVicente-Saez R, Martinez-Fuentes C: Open Science now: A systematic literature review for an integrated definition. J. Bus. Res. 2018; 88: 428–436. Publisher Full Text Reference Source\n\nWilkinson MD, Dumontier M, Aalbersberg IJ, et al.: The FAIR Guiding Principles for scientific data management and stewardship. Sci. Data. 2016; 3(1): 160018. PubMed Abstract | Publisher Full Text Reference Source\n\nYoshioka T, Herman G, Yates J, et al.: Genre taxonomy: A knowledge repository of communicative actions. ACM Trans. Inf. Syst. 2001; 19(4): 431–456. Publisher Full Text Reference Source"
}
|
[
{
"id": "137000",
"date": "19 Jul 2022",
"name": "Guy A. Rouleau",
"expertise": [
"Reviewer Expertise Neuroscience",
"Genetics",
"Neurology",
"Open Science"
],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis article is about the development of a program created by the Carnegie Mellon University Libraries to support Open Science practice at CMU, in particular by offering educational and support services around various platforms.\nA good paper describing an important initiative. Their development of a unified program, as well as the generation of useful metrics, are an important contribution to the development of Open Science at institutions.\nAs discussed in the comments below, there is some information they could add about the history of open science/open research that would add clarity to the text, as well as a few minor revisions that should be made to supply necessary information in figures.\n1. Greater discussion of the historical role of libraries in open research practices.\nIn the introduction the authors use the sentence “As Open Science has matured, academic libraries have also entered this space leveraging the natural alignment with existing services and principles related to information access and dissemination.” This sentence somewhat mischaracterizes the historical role of libraries in the Open Science movement. To a significant extent open science emerged from the Open Access movement, and libraries have been heavily involved in that movement since at least 1997 when the Association of Research Libraries founded Scholarly Publishing and Academic Resources Coalition (SPARC). It would be more accurate to say that the role of libraries has always been important to the movement to open science, but that they have more recently started to play an instrumental role beyond open access to publications.\n2. Earlier introduction to the relationship between reproducibility and open science.\nThe authors introduce the concept of reproducibility on page 5, both in the google search string they used to quality control their search of peer institutions and in the first sentence in the section on “Program Implementation.” To readers versed in Open Science this connection will likely come as no surprise, but to the less versed reader the reference to reproducibility comes somewhat out of the blue. Why, for example, should that be included in a search for Open Science relevant programs? A brief explanation, perhaps in the introduction near where they address the “five schools of thought” would make their search strategy and the other references to reproducibility clearer. This observation is also relevant to the comment below on the history of the field of psychology and its relevance to the development of Open Science.\n3. Further historical context around the field of psychology.\nSimilar to the comment on the history of libraries, and related to the comment on reproducibility, there are several ways a brief discussion of the history of the relationship between psychology and open science would add clarity to the paper. Unlike with libraries’ advocacy for Open Access, which fed into Open Science and which were primarily concerned with rising subscription fees and the efficient dissemination of knowledge, psychology researchers have been a major force in the history of Open Science primarily as an aid to reproducibility and replicability.\nAdding some content about this history would help both to make clear the relationship between reproducibility/replicability and open science—indeed psychologists were the first to identify it as a crisis (https://journals.sagepub.com/doi/10.1177/1745691612465253) – but would also contribute to why the CMU Psychology department has such a large number of users (see Fig. 3).\n4. Adding some discussion of the difficulties in sharing sensitive human data and what this might imply for the data collected.\nWhile the authors address the difficulties posed by ethical constrains on sharing sensitive human data in the section “Discussion and future directions”, acknowledging this particular difficulty in earlier sections where they discuss the data around departmental users would add important interpretive context for why departments that deal with this kind of data regularly (for example the Centre for the Neural Basis of Cognition) might show fewer users.\n5. Acronyms used in Figure 3.\nThe “EPP” acronym on the x-axis in Figure 3 is not explained in the text below the figure, though it likely refers to the department of Engineering and Public Policy. Also, the use of “iii” rather than “III” for the Integrated Innovation Institute isn’t consistent with the capitalization of the other acronyms, though there may be a reason for this I am not aware of.\n6. Note on important policy changes that may impact metrics\nWhile it may not be possible in the context of this paper, it would be useful in future work for the authors to develop a way of assessing important events at the institutional, state, and national levels that impact the uptake of Open Science practices. The emergence of, for example, the NIH policy on data management and sharing may have a significant impact on their metrics in the absence of activity by the OSDC itself. They do address this to some extent in the proposed discussions with “superusers” in the section “Applications of the logic model and 5W1H framework”, and it will be very difficult to give a fully causal account, but at least acknowledging this confound further, or creating some automated system through google alerts or scraping relevant twitter hashtags/keywords (e.g., “Open Science” + “Policy” + filtering for CMU, state, and/or national users) could help provide such a timeline. If, for example, they see an increase in some metrics in the absence of events in the timeline, they can be more certain that the effect is endogenous.\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? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate? Yes\n\nAre all the source data underlying the results available to ensure full reproducibility? Partly\n\nAre the conclusions drawn adequately supported by the results? Yes\n\nIs the case presented with sufficient detail to be useful for teaching or other practitioners? Yes",
"responses": [
{
"c_id": "9080",
"date": "09 Jan 2023",
"name": "Huajin Wang",
"role": "Author Response",
"response": "We thank Drs. Rouleau and Roskams-Edris for your thoughtful comments and suggestions. Please see our point-by-point response below. 1. \"Greater discussion of the historical role of libraries in open research practices. In the introduction the authors use the sentence “As Open Science has matured, academic libraries have also entered this space leveraging the natural alignment with existing services and principles related to information access and dissemination.” This sentence somewhat mischaracterizes the historical role of libraries in the Open Science movement. To a significant extent open science emerged from the Open Access movement, and libraries have been heavily involved in that movement since at least 1997 when the Association of Research Libraries founded Scholarly Publishing and Academic Resources Coalition (SPARC). It would be more accurate to say that the role of libraries has always been important to the movement to open science, but that they have more recently started to play an instrumental role beyond open access to publications.\" Thank you for this insightful comment. We agree that the true characterization of libraries in the open science movement is more significant than previously stated. We have added a paragraph to the Introduction that discusses SPARC and the role of libraries and the open access movement as an early catalyst for open science. 2. \"Earlier introduction to the relationship between reproducibility and open science. The authors introduce the concept of reproducibility on page 5, both in the google search string they used to quality control their search of peer institutions and in the first sentence in the section on “Program Implementation.” To readers versed in Open Science this connection will likely come as no surprise, but to the less versed reader the reference to reproducibility comes somewhat out of the blue. Why, for example, should that be included in a search for Open Science relevant programs? A brief explanation, perhaps in the introduction near where they address the “five schools of thought” would make their search strategy and the other references to reproducibility clearer. This observation is also relevant to the comment below on the history of the field of psychology and its relevance to the development of Open Science.\" We have added a separate paragraph to address this and the following comment. The paragraph introduces the reproducibility crisis and highlights its role in facilitating new practices of open science. 3. \"Further historical context around the field of psychology. Similar to the comment on the history of libraries, and related to the comment on reproducibility, there are several ways a brief discussion of the history of the relationship between psychology and open science would add clarity to the paper. Unlike with libraries’ advocacy for Open Access, which fed into Open Science and which were primarily concerned with rising subscription fees and the efficient dissemination of knowledge, psychology researchers have been a major force in the history of Open Science primarily as an aid to reproducibility and replicability. Adding some content about this history would help both to make clear the relationship between reproducibility/replicability and open science—indeed psychologists were the first to identify it as a crisis (https://journals.sagepub.com/doi/10.1177/1745691612465253) – but would also contribute to why the CMU Psychology department has such a large number of users (see Fig. 3).\" See response above for Item 2, which also addresses this comment. 4. \"Adding some discussion of the difficulties in sharing sensitive human data and what this might imply for the data collected. While the authors address the difficulties posed by ethical constrains on sharing sensitive human data in the section “Discussion and future directions”, acknowledging this particular difficulty in earlier sections where they discuss the data around departmental users would add important interpretive context for why departments that deal with this kind of data regularly (for example the Centre for the Neural Basis of Cognition) might show fewer users.\" We have no evidence that ethical constraints are a factor that CNBC has fewer users. We think it’s more likely due to the smaller department size. We’d like to eventually use proportion to represent user size in each department but at the moment we do not have data on department sizes. See also the response to Reviewer #2. 5. \"Acronyms used in Figure 3. The “EPP” acronym on the x-axis in Figure 3 is not explained in the text below the figure, though it likely refers to the department of Engineering and Public Policy. Also, the use of “iii” rather than “III” for the Integrated Innovation Institute isn’t consistent with the capitalization of the other acronyms, though there may be a reason for this I am not aware of.\" Thanks for pointing out the errors in acronyms. “EPP” indeed refers to Department of Engineering and Public Policy. This has now been added in the figure legend. As for the acronym of Integrated Innovation Institute (iii), lowercase letters were intentionally used by the department, presumably as a design choice. 6. \"Note on important policy changes that may impact metrics. While it may not be possible in the context of this paper, it would be useful in future work for the authors to develop a way of assessing important events at the institutional, state, and national levels that impact the uptake of Open Science practices. The emergence of, for example, the NIH policy on data management and sharing may have a significant impact on their metrics in the absence of activity by the OSDC itself. They do address this to some extent in the proposed discussions with “superusers” in the section “Applications of the logic model and 5W1H framework”, and it will be very difficult to give a fully causal account, but at least acknowledging this confound further, or creating some automated system through google alerts or scraping relevant twitter hashtags/keywords (e.g., “Open Science” + “Policy” + filtering for CMU, state, and/or national users) could help provide such a timeline. If, for example, they see an increase in some metrics in the absence of events in the timeline, they can be more certain that the effect is endogenous.\" It’s a great point by the reviewer that in order to draw a conclusion on causal effect, one would need to factor in major confounding factors, such as external policy changes. We think that adding a “date” field across our data collection process will help to address this issue as it would enable us to associate changes in usage with external influencing factors. The lack of a “date” field was addressed in Limitations of current data sources and future data management strategy section; we now added a sentence to specifically draw attention to confounding from external signals."
}
]
},
{
"id": "141852",
"date": "22 Jul 2022",
"name": "Verena Heise",
"expertise": [
"Reviewer Expertise Open Science",
"Reproducibility",
"Meta-research",
"Biomedical 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\nThe article introduces an Open Science program developed at Carnegie Mellon University Libraries that supports researchers with tools, services and training to develop open and reproducible workflows. Additionally, it describes a framework for assessing the success of this program. Both of these aspects are very interesting for other institutions that are engaged in promoting open and reproducible research and would like to or are in the process of setting up similar services.\nThe article is very informative but nevertheless I have a couple of major and minor comments that I hope will be helpful to the authors:\nMajor items\nThe \"end-to-end\" model In several places, the program is described as an end-to-end model for research projects and I wonder whether a slightly more cautious phrasing might be more appropriate. The program does offer very useful services across the stages of a research project but a) these services are not necessarily applicable to all types of research and only cover the \"ends\" of specific types of research, e.g. those that deal with mostly digital data, and b) an important stage of a research project is missing: the one where we develop hypotheses, e.g. through literature reviews. The latter might be part of the services offered but it is currently not mentioned in the manuscript.\n\nCitizen Science This is mentioned in a couple of places (e.g. Figure 1) as a service supported by the OSDC (Open Science & Data Collaborations) but there is very little information on it in the paper. Could you clarify what the role of OSDC is with respect to Citizen Science? And just out of curiosity: Citizen Science can be included in research projects at all stages, so I was wondering why it only seems to be applicable to the data collection and analysis stage in Figure 1?\n\nFigure 1 I was wondering whether it might make sense to remove the grey boxes since the article mostly focuses on the OSDC. Two items that are not currently explained in the legend or the following table are the DMPTool and OpenRefine, so it would be great to add some more info on these.\n\nTable 2 It would be quite helpful to explain the categories in the table in a bit more detail. For example, what is the difference between Library sponsored OS programs and Library Open Research programming?\n\nFigure 2 Activities: I’m not sure what the Emerald Cloud Lab integration and Reproducibility MiniSeries refer to. Outputs: I guess the training sessions are related to the tools themselves? Would it make sense to have these in the second category together with the other workshops? Weekly office hours: I was wondering why this is relevant. I guess the question would be how many people actually come to get support during the office hours? Outcomes (this is a conceptual point, so does not necessarily need to be addressed for this paper): I’m surprised they are defined as outcomes for users, not the OSDC. This might just be the way the strategic goals are defined for OSDC and it’s absolutely fine if the figure reflects these. I wonder whether that’s the best way to define outcomes for the OSDC because it's probably difficult to assess whether the OSDC has been successful with that selection of outcomes, especially the medium and long-term ones. For example, shifts in research culture require much more than just availability of training, tools and services, so it's not only down to the OSDC whether those cultural shifts actually materialise.\n\nFigure 3 As far as I can see the acronyms EPP, CIT, CYLAB are not covered in the figure legend. And another conceptual comment that does not necessarily need to be addressed here: it might be interesting to look at user numbers in relation to size of the institutes, e.g. percentage of faculty using services, so that institutes of different sizes can be compared quite easily.\n\nFigure 5 This is a very stupid question but it would be great to clarify that these are new items/ new registrations per year and not the total numbers (e.g. of accounts) in that year, which would be the sum of accounts from the previous year plus new accounts?\nMinor items\nIntroduction\n“This trend has been a response to changes in the funding and publishing landscape, the nature of research collaboration, the emergence of digital research infrastructures and cultural shifts in scientific practice” - I wonder whether a couple of references might help here or a some examples of the changes this refers to. I guess it refers to changes such as open access/ open data mandates but it would be helpful to see what the authors mean.\n\nDefinition of Open Science: I wonder whether it might be helpful to include the definition used in the UNESCO recommendation (https://en.unesco.org/science-sustainable-future/open-science/recommendation) because it's a definition of Open Science that is gaining quite a bit of traction, especially among policy makers.\nDiscussion\nI was wondering why the first paragraph is mostly about data sharing and does not cover Open Science practices more broadly since OSDC is about more than just data sharing.\n\nThe sentence that ends with “prevents us from being able to make a clear value proposition to researchers for whom productivity, efficiency, and impact are the most important factors” might need rephrasing. I would hope that the impact of reproducible workflows and data management are obvious to many researchers because they have clear impacts on efficiency and impact (e.g. see higher citation rates of openly available papers and data). I understand that it can be difficult to quantify some of those aspects but nevertheless there is a clear value proposition for using open/ reproducible workflows and a number of papers have dealt with the selfish reasons for working in a more open/ reproducible way (e.g. McKiernan paper cited already and https://genomebiology.biomedcentral.com/articles/10.1186/s13059-015-0850-7)\n\nThere are a couple of sentences about data management, e.g. this one: \"the current data and the 5W1H metrics framework will serve as a baseline to develop a strategy for data management in the future to guide data collection, update, and analysis”. I think this refers to management of user data related to services and not research data itself and it would be great if that could be rephrased slightly to make the distinction more obvious.\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? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate? Yes\n\nAre all the source data underlying the results available to ensure full reproducibility? Partly\n\nAre the conclusions drawn adequately supported by the results? Yes\n\nIs the case presented with sufficient detail to be useful for teaching or other practitioners? Yes",
"responses": [
{
"c_id": "9081",
"date": "09 Jan 2023",
"name": "Huajin Wang",
"role": "Author Response",
"response": "Thank you Dr. Verena Heise for your thorough review of our work. Please see our point-by-point response below. Major items 1. \"The \"end-to-end\" model. In several places, the program is described as an end-to-end model for research projects and I wonder whether a slightly more cautious phrasing might be more appropriate. The program does offer very useful services across the stages of a research project but a) these services are not necessarily applicable to all types of research and only cover the \"ends\" of specific types of research, e.g. those that deal with mostly digital data, and b) an important stage of a research project is missing: the one where we develop hypotheses, e.g. through literature reviews. The latter might be part of the services offered but it is currently not mentioned in the manuscript.\" We use “end-to-end” to describe a service model for open science with the intention of serving all stages of the research life cycle, but the implementation is a gradual process and the current composition reflects the demands we see at Carnegie Mellon and the tools and expertise that are readily available at the moment. We thank the reviewer for pointing out non-digital data and literature reviews as gaps in our service and will keep them in mind as our program matures. CMU Libraries indeed provide systematic review and evidence synthesis services to help researchers develop hypotheses from literature and increase research rigor and reproducibility, independent from the OSDC program. It would be important to think about how it overlaps and interacts with the OSDC program going forward. 2. \"Citizen Science. This is mentioned in a couple of places (e.g. Figure 1) as a service supported by the OSDC (Open Science & Data Collaborations) but there is very little information on it in the paper. Could you clarify what the role of OSDC is with respect to Citizen Science? And just out of curiosity: Citizen Science can be included in research projects at all stages, so I was wondering why it only seems to be applicable to the data collection and analysis stage in Figure 1?\" Even though citizen science is a direction we aspire to support in the future, we have only started to explore how to support it with our services and so far have only offered a few workshops. In the revised manuscript we have now removed citizen science from Figure 1 and Figure 2 to avoid confusion. 3. \"Figure 1. I was wondering whether it might make sense to remove the grey boxes since the article mostly focuses on the OSDC. Two items that are not currently explained in the legend or the following table are the DMPTool and OpenRefine, so it would be great to add some more info on these.\" We feel that keeping services that are within broader open science but not administered by OSDC in the gray boxes would help readers understand the interaction and relationship between related services in a larger context, as different libraries or universities may structure them differently. We now added additional text to explain this intention (page 3, 3rd paragraph). In addition, we have defined DMPTool and OpenRefine in the figure legend of Figure 1. 4. \"Table 2. It would be quite helpful to explain the categories in the table in a bit more detail. For example, what is the difference between Library sponsored OS programs and Library Open Research programming?\" We have added more information to the Table 2 legend to define the column names more clearly. 5. \"Figure 2. Activities: I’m not sure what the Emerald Cloud Lab integration and Reproducibility MiniSeries refer to.\" We have added to Figure Legend to briefly describe Emerald Cloud Lab and Reproducibility MiniSeries. \"Outputs: I guess the training sessions are related to the tools themselves? Would it make sense to have these in the second category together with the other workshops?\" We’d like to keep tool-specific trainings separate from general skill-building workshops because these activities are led by different teams and often attract different audiences. \"Weekly office hours: I was wondering why this is relevant. I guess the question would be how many people actually come to get support during the office hours?\" We offer weekly office hours to support researchers who have data related questions. The reviewer brings out a great question about participation, unfortunately we haven’t systematically tracked the number of participants especially during the pandemic when office hours become online. We will start tracking these numbers in the next iteration. \"Outcomes (this is a conceptual point, so does not necessarily need to be addressed for this paper): I’m surprised they are defined as outcomes for users, not the OSDC. This might just be the way the strategic goals are defined for OSDC and it’s absolutely fine if the figure reflects these. I wonder whether that’s the best way to define outcomes for the OSDC because it's probably difficult to assess whether the OSDC has been successful with that selection of outcomes, especially the medium and long-term ones. For example, shifts in research culture require much more than just availability of training, tools and services, so it's not only down to the OSDC whether those cultural shifts actually materialise.\" Thanks for asking this conceptual question. We choose to use user behavior to define our outcomes because the goal of the OSDC program is to drive behavior change in researchers. 6. \"Figure 3. As far as I can see the acronyms EPP, CIT, CYLAB are not covered in the figure legend. And another conceptual comment that does not necessarily need to be addressed here: it might be interesting to look at user numbers in relation to size of the institutes, e.g. percentage of faculty using services, so that institutes of different sizes can be compared quite easily.\" The acronyms have now been defined in the Figure Legend. We thank the reviewer for suggesting using proportion to represent usage to normalize for department size. This is a great suggestion and would be something we’d like to implement in the future. However, we don’t have data on the total number of students and faculty in each department at the moment. Additionally, our users are not only faculty but also staff, students, and postdocs, making it even harder to obtain an accurate total number per department. 7. \"Figure 5. This is a very stupid question but it would be great to clarify that these are new items/ new registrations per year and not the total numbers (e.g. of accounts) in that year, which would be the sum of accounts from the previous year plus new accounts?\" The values presented in Figure 5 are cumulative numbers, i.e., the sum of accounts from the previous year plus new accounts. This is now clarified in the figure legend. Minor Items Introduction: 1. \"\"This trend has been a response to changes in the funding and publishing landscape, the nature of research collaboration, the emergence of digital research infrastructures and cultural shifts in scientific practice” - I wonder whether a couple of references might help here or a some examples of the changes this refers to. I guess it refers to changes such as open access/ open data mandates but it would be helpful to see what the authors mean.\" Thank you for this suggestion. We have added parentheticals with examples of each of the changes noted in the sentence along with the following references. Kozlov, M. (2022). NIH issues a seismic mandate: Share data publicly. Nature, 602(7898), 558–559. https://doi.org/10.1038/d41586-022-00402-1 Davidson LA. The End of Print: Digitization and Its Consequence—Revolutionary Changes in Scholarly and Social Communication and in Scientific Research. International Journal of Toxicology. 2005;24(1):25-34. doi:10.1080/10915810590921351 Ponte, D., Mierzejewska, B.I. & Klein, S. The transformation of the academic publishing market: multiple perspectives on innovation. Electron Markets 27, 97–100 (2017). https://doi.org/10.1007/s12525-017-0250-9 Heller, L., The, R., Bartling, S. (2014). Dynamic Publication Formats and Collaborative Authoring. In: Bartling, S., Friesike, S. (eds) Opening Science. Springer, Cham. https://doi.org/10.1007/978-3-319-00026-8_13 Fyfe, A., Coate, K., Curry, S., Lawson, S., Moxham, N., & Røstvik, C. M. (2017). Untangling academic publishing: A history of the relationship between commercial interests, academic prestige and the circulation of research. Cummings, J. N., & Kiesler, S. (2014). Organization theory and the changing nature of science. Journal of Organization Design, 3(3), 1-16. Huizingh, E. K. R. E. (2011). Open innovation: State of the art and future perspectives. Technovation, 31(1), 2–9. https://doi.org/10.1016/j.technovation.2010.10.002 2. \"Definition of Open Science: I wonder whether it might be helpful to include the definition used in the UNESCO recommendation (https://en.unesco.org/science-sustainable-future/open-science/recommendation) because it's a definition of Open Science that is gaining quite a bit of traction, especially among policy makers.\" We have added the UNESCO definition to the text. Discussion 1. \"I was wondering why the first paragraph is mostly about data sharing and does not cover Open Science practices more broadly since OSDC is about more than just data sharing.\" Thanks for pointing out the terminology. We intend to use the term “data” broadly to refer to all research outputs, including data, code, workflow, and more. We added a sentence in the first paragraph of the Discussion and future directions section to clarify. 2. \"The sentence that ends with “prevents us from being able to make a clear value proposition to researchers for whom productivity, efficiency, and impact are the most important factors” might need rephrasing. I would hope that the impact of reproducible workflows and data management are obvious to many researchers because they have clear impacts on efficiency and impact (e.g. see higher citation rates of openly available papers and data). I understand that it can be difficult to quantify some of those aspects but nevertheless there is a clear value proposition for using open/ reproducible workflows and a number of papers have dealt with the selfish reasons for working in a more open/ reproducible way (e.g. McKiernan paper cited already and https://genomebiology.biomedcentral.com/articles/10.1186/s13059-015-0850-7)\" Even though the value of using open/ reproducible workflows have been shown to benefit researchers from a “selfish” perspective, many researchers are still unconvinced or unaware, and the mainstream research culture remains relying on quantifiable metrics to evaluate productivity and impact. We have revised the sentence that the reviewer refers to clarify our view on the value proposition. 3. \"There are a couple of sentences about data management, e.g. this one: \"the current data and the 5W1H metrics framework will serve as a baseline to develop a strategy for data management in the future to guide data collection, update, and analysis”. I think this refers to management of user data related to services and not research data itself and it would be great if that could be rephrased slightly to make the distinction more obvious.\" We agree with the reviewer that the terminology might be confused with “research data management”. We have now changed it to “user data management”."
}
]
}
] | 1
|
https://f1000research.com/articles/11-501
|
https://f1000research.com/articles/11-1207/v1
|
24 Oct 22
|
{
"type": "Research Article",
"title": "Evaluation of left ventricular systolic function in children with sickle cell anemia: contribution of 2D strain",
"authors": [
"Sarra Chenik",
"Aymen Noamen",
"Abyr Bouslimi",
"Houaida Mahfoudhi",
"Sadok Hannachi",
"Hager Barakizou",
"Islam Mejri",
"Znegui Tasnim",
"Wafa Fehri",
"Aymen Noamen",
"Abyr Bouslimi",
"Houaida Mahfoudhi",
"Sadok Hannachi",
"Hager Barakizou",
"Islam Mejri",
"Znegui Tasnim",
"Wafa Fehri"
],
"abstract": "Background: Cardiovascular involvement is not well studied in children with sickle cell disease. The aim of this study was to evaluate the echocardiographic parameters and to investigate speckle tracking echocardiography (STE) interest in detecting subclinical myocardial impairment of children with sickle cell disease. Methods: The study was directed in the echocardiographic laboratory in the military hospital of Tunis between July 2018 and December 2018. 30 patients with sickle cell anemia (SCA) and 30 controls were compared. The echocardiographic measurements were indexed according to body surface. Cardiac output, left ventricular ejection fraction, wall thickness, as well as LV 2-D longitudinal systolic strain were assessed. Results:\nThe SCA Group included 30 patients (11.8 ± 2yrs, sex ratio: 1.31) with homozygous SCA and the C Group included 30 healthy controls (12.7 ± 1,2yrs, sex ratio: 1.27). According to the findings, SCA Group showed significantly larger LV diameter (36.2±2.5mm/m2 vs 29.3±1.3mm/m2, p=0.005). SCA Group also showed lower LV ejection fraction (62%±0.5 vs 65%±5, p=0.001). No significant difference was observed for cardiac output (p=0.4). Otherwise, two-dimensional longitudinal strain of LV was higher in SCA group (-21%±3.07 vs -25%±2.98; p<0.01). Conclusions: Our study highlights several cardiac abnormalities in children with SCA, which could represent a marker of disease severity and point out the importance of the cardiologic screening of these patients.",
"keywords": [
"sickle cell anemia",
"heart disease",
"echocardiography",
"Speckle tracking echocardiography. Global longitudinal strain",
"Left ventricular systolic function",
"child."
],
"content": "List of abreviations\n\n2D: two-dimensional\n\nC: Control\n\nDTI: Pulsed Doppler tissue\n\nGLS: global longitudinal strain\n\nIVST: interventricular septal wall thickness\n\nLV: Left ventricular\n\nLVEDD: Left ventricular end-diastolic diameter\n\nLVEF: Left ventricular ejection fraction\n\nLVESD: Left ventricular end-systolic diameter\n\nLVGLS: Left ventricular global longitudinal strain\n\nLVM: Left ventricular mass\n\nMAPSE: mitral annular plane systolic excursion\n\nPWT: Posterior wall thickness\n\nS’: systolic mitral annulus velocity\n\nSCA: Sickle cell anemia\n\nTM: Time Movement\n\nTTE: transthoracic echocardiography\n\n\nIntroduction\n\nSickle cell disease or sickle cell anemia is an autosomal recessive genetic disease linked to a hemoglobin abnormality leading to the deformation of red blood cells.\n\nThe disease affects more than 50 million people worldwide, particularly in black Africa and the Mediterranean region.1 Globally, hemoglobin disorders are responsible for about 3.4% of deaths in children under 5 years of age.1,2\n\nNevertheless, the life expectancy of sickle cell patients has improved in recent years with healthier lifestyles, medical monitoring, and new therapies. The increase in their life expectancy has led to the appearance of chronic multi-systemic complications in addition to acute complications. Indeed, despite a common genetic basis and a similar physiopathology, sickle cell patients have a clinical phenotype of very variable severity.\n\nIt is therefore a serious chronic disease whose prognosis depends on the occurrence of complications, particularly cardiovascular ones. However, the discovery of this cardiovascular disorder is often made in adulthood, and the literature is poor on its detection in childhood, probably because of the lack of systematic cardiological follow-up in these children.\n\nNowadays, with the advent of new echocardiographic techniques such as two-dimensional (2D) strain, it has been possible to demonstrate systolic and diastolic dysfunction earlier than with conventional echocardiography.\n\nThe advent of 2D strain has enabled early detection of cardiac damage in many previously unrecognized chronic conditions and is increasingly a prognostic marker to guide patient management.\n\nIt is a new technique that studies myocardial motion by tracking speckles, which are acoustic markers of the myocardium. Local tissue motion is represented by the geometric displacement of each speckle. Several software packages have been developed to allow temporal and spatial processing of the image obtained by the 2D strain.\n\nThus, we undertook this study of sonographic aspects of the hearts of children with sickle cell disease, to identify the different possible abnormalities detected in the left ventricular function by conventional ultrasound and the 2D strain technique in order to assess cardiac injury and establish a clinical and echocardiographic monitoring scheme.\n\nThe main objective of our study is to evaluate the interest of 2D strain in the detection of left ventricular (LV) myocardial damage at a sub-clinical stage in children with sickle cell disease.\n\n\nMethods\n\nThis is a descriptive cross-sectional study conducted in the cardiology department at the Main Military Hospital of Instructions in Tunis which took place over six months between July 2018 and December 2018.\n\nSample size calculation\n\nWe estimated that 2.4% of patients were followed up at the pediatric department and referred to pediatric cardiology consultation during the study period. A sample size of 35 patients would be required to achieve statistical significance (power: 0.8; alpha: 0.05) calculated using a predictive formula.3\n\nInclusion criteria of the SCA group\n\nWe included first the SCA group comprising 30 children followed up at the pediatric department of the Tunis military hospital and who benefited from transthoracic echocardiography (TTE) during their follow-up.\n\nNon-inclusion criteria of the SCA group\n\nWe did not include patients with congenital heart disease, associated valvular heart disease, or those who had undergone cardiac surgery (n=1). We also did not include two patients who were lost to follow-up at the time of the study and one participant who requested to withdraw from the study.\n\nExclusion criteria\n\nExcluded from this study were patients with poor echogenicity during TTE (n-=).\n\nControl group\n\nOur study included a second group “C” of 30 controls free of any cardiovascular or respiratory pathology and not presenting anemia at the time of the study.\n\nThese were children who had been hospitalized in the pediatric department and referred to a pediatric cardiology consultation for the assessment of a heart murmur or for the exploration of atypical chest pain, and who had a normal echocardiography.\n\nThe control group was then matched according to age, gender and body mass index with the SCA group.\n\nThe participants were contacted by phone and were identified by name and date of birth, and then anonymized during data entry. Data collection was carried out by a single physician using a patient information sheet. All parents seemed to find participation in the study beneficial.\n\nThey agreed to participate, and their children underwent an echocardiographic examination because they found it non-invasive and important for disease assessment.\n\nClinical and paraclinical data\n\nData collected were age, gender, and medical history. A study of the medical records of sickle cell patients was carried out and allowed us to collect clinical and paraclinical data such as: age of discovery of the disease, baseline hemoglobin level, rate of transfusion, existence of complications and treatments received.\n\nEchocardiography\n\nAll study subjects from both SCA and control groups underwent echocardiographic examination and speckle tracking measurement, all performed by a single experienced cardiologist who was blinded to all clinical data.\n\nThis examination was performed using a vivid E7 ultrasound system (GE healthcare, Horten, Norway) with an S 5-1 probe according to the guidelines of the American Society of Echocardiography and the European Association of Cardiovascular Imaging (12). Measurements were performed in the parasternal long axis, parasternal short axis, apical two, three, four, five cavities and subcostal. During the examination the scale was set between 12 and 20 cm/s to avoid spectral folding.\n\nEach incidence was taken so that the angle between the myocardial wall under study and the ultrasound beam does not exceed 30 degrees. All echographic data was then stored on a central memory unit, allowing for the post-processing and adjustment of measurements, including measurement work on pulsed Doppler tissue (DTI) spectra by averaging the results of three to five cardiac cycles.\n\nLeft ventricular study\n\nLeft ventricular end-diastolic diameter (LVEDD), left ventricular end-systolic diameter (LVESD), interventricular septal wall thickness (IVST) and posterior wall thickness (PWT) were measured with 2D targeted M-mode tracing. Left ventricular mass (LVM) was estimated using Devereux formula.\n\nLeft ventricular ejection fraction (LVEF) was calculated using Simpson’s biplane method of discs then systolic mitral annulus velocity (S’) was assessed using tissue Doppler imaging in 2D mode of the mitral annulus in the four-chamber view.\n\nStrain analysis by speckle-tracking echocardiography: myocardial deformation study\n\nSpeckle tracking analysis was conducted offline on the apical four chamber, apical long axis, and apical two chamber views that were previously stored in Digital Imaging and Communications in Medicine (DICOM) format. Analysis of echocardiographic images was performed offline using Echo PAC (GE Medical Systems, Norway).\n\nThree cardiac cycles were recorded in cine loop format at a frame rate between 50 and 80 frames per second as two-dimensional grayscale views.\n\nOne end systolic frame is selected by the operator to perform the manual tracing of the endocardial border of the left ventricle (LV), this tracing is then used by the software to create a region of interest. The myocardium is then automatically divided in segments according to the standard 16-segment model of the LV.4\n\nThe quality of myocardial tracking was visually checked in real time then manually corrected to ensure optimal tracking. The software then tracks the deformation of the myocardium during cardiac cycle to calculate peak systolic segmental strain. Global longitudinal strain (GLS) is determined as the average of the segmental strains (Figures 1, 2).\n\nAll statistical analyses were conducted using SPSS 25.0 software (IBM SPSS Inc., Chicago, Illinois, USA). Quantitative variables were tested for normal distribution using the Shapiro-Wilk and Kolmogorov-Smirnov tests then expressed as mean±standard deviation. Qualitative variables were expressed as counts and percentages.\n\nParametric data was then compared using Student’s T test for independent-samples and non-parametric data was compared using the Mann-Whitney-Wilcoxon test.\n\nPotential relation between clinical or echocardiographic characteristics of SCA patients on GLS measurement was assessed using chi-square or Fisher’s exact test for qualitative data and Pearson’s correlation coefficient and Spearman’s rank correlation coefficient for quantitative data. All statistical tests were two-sided, and the level of statistical significance was set up at p<0.05.\n\nBibliographic research was undertaken on PubMed through MESH research using keywords: sickle cell anemia; heart disease; echocardiography; Speckle tracking echocardiography. Global longitudinal strain; Left ventricular systolic function; child. We include publications in French and English between 19270 and 2020.\n\nAfter approval from the ethics committee of the main military hospital of Tunis (local committee for the protection of persons of the Military Hospital of Tunis (N°65/2022/CLPP/Military Hospital of Tunis) we included children under 18 years of age, regularly followed in the pediatric department, with homozygous SCA. For logistical reasons, the number of patients was limited to 30. Written informed consent was then obtained from the parents in view of the non-invasive nature of the parameters studied. Anonymity was respected during data entry.\n\n\nResults\n\nDemographics\n\nThe mean age of our population was 12 ±4 years with extremes ranging from 3 to 17 years. The mean age of discovery of SCA was 2 years. Our population included 13 girls, 43.3% of the population, and 17 boys (56.7%). The average weight in our population was 35±14 kg, with an average height of 131±31 cm, or an average body surface area of 1.16±0.32 m2.\n\nClinical data\n\nClinical examination data\n\nBlood pressure measurement found a mean systolic blood pressure of 103±9 mmHg and a mean diastolic blood pressure of 68±9 mmHg. The mean heart rate was 93±8 cpm.\n\nClinical symptomatology and complications\n\nThe symptoms found in the SCA group and the complications reported are summarized in Table 1.\n\n* ATS=Acute Thoracic Syndrome.\n\nBiological data\n\nThe average hemoglobin level in our patients was 8.6±0.5 g/dl. The mean ferritin level was 824±32 μg/l for a normal value between 7 and 140 μg/l.\n\nPharmacological treatment\n\nThe various medications received by our patients were penicillin V in 43%, hydroxycarbamin in 46%, deferoxamine in 16% and vitamin E in 96%. All patients were on folic acid.\n\nClinical and anthropometric data\n\nThe comparison of the age of the children in the two groups did not show a statistically significant difference. The mean age in the SCA group was 12 years compared to 11 years in the C group (p=0.235). The sex ratio was 0.76 in the SCA group with a female predominance. In the C group, there was a male predominance with a sex ratio of 1.5 but this difference was not statistically significant (p=0.150).\n\nAnthropometric data were comparable in both groups as shown in Table 2.\n\nStandard echocardiography data\n\nMorphological study of the left ventricle\n\nThe dimensions of the left ventricle in sickle cell children are summarized in Table 3.\n\n* LVtdD=telediastolic diameter of the left ventricle.\n\n† ISW=interseptal wall.\n\n‡ LVMind=indexed left ventricular mass.\n\n§ RWT=Wall Relative Thickness.\n\nStudy of the LV systolic function\n\nWe measured the left ventricular ejection fraction (LVEF) by the Teicholtz method in both groups of patients. In SCA group, the mean LVEF was 58% with a minimum LVEF of 52% and a maximum of 77%. LVEF was maintained in all sickle cell patients. In time movement (TM) mode, we found a mitral annular plane systolic excursion (MAPSE of 14.6±3 mm in SCA group versus 13±3.3 mm in C group. In the LV tissue Doppler study, an LV S’ wave was measured at 9±2.6 cm/s in SCA group versus 8.7±1.7 cm/s in C group (p=0.685). The calculation of indexed cardiac output was comparable in both groups (Table 4)\n\n* LVEF=left ventricular ejection fraction.\n\n‡ MAPSE=Mitral annular plane systolic excursion.\n\nStudy of longitudinal myocardial deformation using the speckle tracking technique\n\nThe measurement of the left ventricular global longitudinal strain LVGLS showed a mean value of -21.2±3% in the SCA group.\n\nThe GLS was impaired in 46% of cases for a reference threshold of -20%.\n\nThe comparative study between the two groups of patients showed a statistically significant difference with a mean value in SCA group of -21.22±3%, compared to -25.03±2.9% in C group (p<0.01) (Figure 3).\n\nPredictive factors for alteration of the global longitudinal strain of the LV\n\nWe found a p-value in the limit of significance between the occurrence of certain complications and the alteration of GLS such as osteonecrosis (p=0.051), splenomegaly (p=0.051, R=0.61). The occurrence of splenic sequestration (p=0.07, R=0.34) and dyspnea (p=0.075, R=0.38) were close to statistical significance. The influence of hydroxycarbamine treatment on GLS alteration was close to significance (p=0.075, R=-0.07). We found a statistically significant influence between GLS alteration and hydroxycarbamine dosage (p=0.043) (Table 5).\n\n‡ VOC=vaso-occlusive crisis.\n\n* ATS=acute chest syndrome.\n\nBiological data\n\nWe did not find any association between the alteration of the GLS and the biological data: hemoglobin level (p=0.261, r=-0.11), Ferritin level (p=0.734, r=-0.08).\n\nEchocardiographic parameters\n\nLV dilatation was correlated with impaired LV GLS (p=0.04; R=0.27). Furthermore, LV hypertrophy was also associated with Strain impairment with a statistically significant p for both indexed LV mass (p=0.007; r=0.41) and posterior wall thickness (p=0.016; R=0.33).\n\n\nDiscussion\n\nWe performed a comparative study in which we were interested in the comparison of several morphological and functional echographic parameters of the left ventricle between a first group (SCA group) of 30 children under 18 years of age followed up for major sickle cell syndrome, and a second group (C group) of 30 healthy controls. The two groups of patients were comparable for sex, age, and body surface area. At the end of the study, we found some morphological differences in the left ventricle between the two groups.\n\nOur results mainly support the use of 2D strain in the assessment of left ventricular function in our population. The study of the myocardial strain by speckle tracking technique allowed us to objectivate a statistically significant difference in the GLS between the two groups of patients (p<0.01) despite a preserved value of the LVEF.\n\nThe analytical study of the data collected allowed us to show a significant correlation between this alteration in strain and certain clinical and echographic parameters.\n\nIn our study, we noted a significantly greater dilation of the LV in the SCA group compared to the C group. Indeed, the chronic anemia due to hemolysis in this disease induces an increase in cardiac output with a minimal increase in heart rate. The systolic ejection volume (SEV) then increases, resulting in significant dilation of the LV.5\n\nIn addition, this chronic intravascular hemolysis is often associated with pulmonary vaso-occlusive events, which contributes to oxygen desaturation of the blood and increased cardiac output to accommodate this hypoxemia.\n\nThe degree of LV dilatation has been described as proportional to the degree of anemia in some studies.6,7 However, increased cardiac output in non-sickle cell anemia usually occurs when the hemoglobin level is less than or equal to 7 g/100 ml. A few studies have been carried out using hemodynamic measurements of cardiac output in homozygous sickle cell disease and have confirmed the existence of a significant increase in resting cardiac output in most patients, even for hemoglobin levels of 9 to 10 g/100 ml. Thus, for the same hemoglobin level, resting cardiac output was higher in sickle cell patients than in subjects with chronic anemia of other etiologies.1 This is probably due to the oxygen desaturation of arterial blood, due to the decreased affinity of hemoglobin S for oxygen, but even more so to the formation of left-sided intra pulmonary shunts due to vaso-occlusive phenomena.8\n\nThis increase in cardiac output has long been incriminated as the primum movens of cardiac damage in sickle cell patients. It has been demonstrated in several studies by measurement of cardiac output by transthoracic ultrasound and by invasive hemodynamic measurements and has often been associated with an increase in morbidity and mortality in these patients.9\n\nIn the course of this work, we noted significantly more left ventricular hypertrophy in the SCA group compared to the C group. This LV remodeling was rather eccentric in our population. Indeed, to compensate for the increase in end-diastolic diameter, and therefore the increase in parietal stress, there is an increase in left ventricular mass. According to Laplace’s law, ventricular wall stress is directly proportional to LV pressure and diameter, and inversely proportional to wall thickness. In addition, iron overload may develop during transfusions and have an impact on the evolution of this myocardial hypertrophy.10\n\nLV systolic function was assessed by several parameters in this study. LVEF measurement by the Teicholtz method was maintained in all patients but the mean was significantly lower in the SCA group. No significant differences were found for the rest of the parameters, as well as for cardiac output.\n\nThis result has been confirmed by several studies and meta-analyses. A meta-analysis published in 2013 included 19 studies involving 841 subjects with sickle cell disease and 554 controls. In this meta-analysis no statistically significant difference was found in LVEF and RF between patients and controls with p values of 0.76 and 0.28 respectively.11\n\nLamers et al. studied 57 children with sickle cell disease compared to 50 healthy children. They studied myocardial contractility using parameters that depend on loading conditions, such as shortening fraction (SF), which was lower in sickle cell children, but within the normal range for age.12\n\nWali et al. studied LV systolic function in 40 children with sickle cell disease compared to 25 controls. No statistically significant differences were reported in the study of LV systolic function.13\n\nThe various studies that have assessed LV systolic function in sickle cell patients have concluded that myocardial contractility is impaired based on parameters independent of loading conditions. The load-dependent measures would be compensated for in early life and would progressively deteriorate with age.14 Consistent with these data, assessment of LV systolic function by measuring MAPSE in TM mode and LV S’ wave velocity by tissue Doppler at the mitral annulus found no significant difference in our pediatric population compared with the control group.\n\nThe mechanism of cardiomyopathy in sickle cell disease is twofold: chronic left ventricular volume overload due to anemia associated with repeated ischemic events due to thrombosis of the coronary microcirculation. This leads to dilation and myocardial remodeling which will evolve towards impairment of LV systolic function. However, the interpretation of the usual systolic function parameters must be done with caution under abnormal loading conditions in sickle cell patients, and the demonstration of this impairment requires ultrasound parameters that are not dependent on loading conditions.\n\nValue of speckle tracking in the study of systolic function in sickle cell patients\n\nIn this work, the LVGLS was significantly more impaired in sickle cell patients than in controls. This result has been found in some studies.13,15\n\nIndeed, global longitudinal strain correlates better with myocardial fibrosis than LVEF and its interest in these patients lies mainly in its sensitivity to detect early alterations in systolic function despite a preserved LVEF.16,17\n\nIndeed, the LV dysfunction reflected by the altered strain in sickle cell patients could be the result of myocardial ischemia, fibrosis or iron deposition in the myocardium or ventricular hypertrophy which could be associated at the beginning with a preserved LVEF value.18 Furthermore, it is recognized that in ischemic or non-ischemic dilated heart disease, alterations in GLS generally correlate well with LVEF and precede it during the course of the disease. This correlation would be particularly useful in practice for monitoring cardiomyopathy in sickle cell patients as it could provide a reproducible estimate of LVEF measured in Simpson biplane and could thus be proposed as a reproducible tool to reduce inter- and intra-observer variability in the study of myocardial strain and thus improve patient management by identifying myocardial damage at a sub-clinical stage.19\n\nAfter analysis of the clinical data in our sickle cell patients, the predictive factor of an alteration of the LVGLS found in this work was mainly the treatment with hydroxycarbamine. A trend towards significance was noted for the occurrence of complications including osteonecrosis, splenomegaly, and splenic sequestration.\n\nIndeed, hydroxycarbamine is a particularly important molecule in the treatment of sickle cell disease by stimulating the synthesis of foetal hemoglobin which plays a protective role in these patients. A study published by Sachdev et al. in 201720 demonstrated a significant improvement in coronary analysis of the ultrasound parameters and revealed a correlation between alteration of LV GLS and LV dilatation and hypertrophy. LV dilatation was correlated with GLS alteration in this work. These echographic findings found in several series21,22 derive from the pathophysiological mechanisms of cardiac involvement in sickle cell disease and could be explained mainly by the progression of myocardial fibrosis and diastolic dysfunction which constitute an independent prognostic factor in these patients.20 The GLS study could thus be proposed as an endpoint for the prospective study and ultrasound follow-up of sickle cell patients, but its clinical consequences remain to be evaluated by large-scale studies.23\n\nRecent studies of the genetic basis of transmission of sickle cell disease have identified certain genotypes associated with cardiovascular disease.24 It is therefore necessary to couple the study of echocardiography and strain with genetic analyses to better study the different phenotypes and forms of this disease, to eventually guide screening, follow-up, and genetic counselling in these patients.\n\nFinally, the exact incidence of cardiomyopathy in sickle cell disease is still unclear.25,26\n\nThe interpretation of our results was further limited by the lack of recognized standard norms of strain in the pediatric population and by the complexity of using the reference values of the Z score, in the morphological study of the LV, which remains little used in current practice, but which may have reservations about our results.\n\nIn this work, the LVGLS was significantly more impaired in sickle cell patients than in controls. However, larger scale studies are therefore required to better study the role of LV strain in the evaluation of myocardial damage in children with sickle cell disease and in the estimation of the risk of progression to heart failure in these patients, in order to guide therapeutic management. It seems necessary to estimate the cardiovascular risk of this population and to propose management algorithms that may include the study of myocardial deformation to reduce the risk of sudden death and the progression to heart failure.\n\n\nConsent\n\nWritten informed consent was obtained from the parents.\n\n\nAuthor contributions\n\nEach author has contributed to this work as follows:\n\nSarra Chenik: Conceptualization, Data Curation, Methodology, Resources, Validation, Writing – Original Draft Preparation, Writing – Review & Editing\n\nAymen Noamen: Data Curation, Methodology, Resources, Validation, Writing – Original Draft Preparation\n\nAbyr Bouslimi: Data Curation, Methodology, Supervision, Validation, Writing – Original Draft Preparation, Writing – Review & Editing\n\nHouaida Mahfoudhi: Supervision, Validation, Visualization, Writing – Review & Editing\n\nSadok Hannachi: Conceptualization, Data Curation, Methodology, Resources, Supervision, Validation, Writing – Original Draft Preparation, Writing – Review & Editing\n\nHager Barakizou: Resources, Validation, Visualization\n\nIslem Mejri: Validation, Visualization, Writing – Review & Editing\n\nTasnim znegui: Visualization\n\nWafa Fehri: Supervision, Validation, Visualization, Writing – Review & Editing",
"appendix": "Data availability\n\nfigshare: Echocardiographic evaluation in children with sickle cell anemia: contribution of 2D strain, https://doi.org/10.6084/m9.figshare.20949031.v4. 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\nOndze-Kafata LI, Sanouiller A, Hedreville M, et al.: Aspects écho-cardiographiques au cours de la drépanocytose en Guadeloupe. Pan Afr. Med. J. 2014; 18: 45.\n\nDahmani F, Benkirane S, Kouzih J, et al.: Profil épidémiologique des hémoglobinopathies: étude autour du cas index: étude transversale descriptive. Pan Afr. Med. J. 2017; 27: 150.\n\nPourhoseingholi MA, Vahedi M, Rahimzadeh M: Sample size calculation in medical studies. Gastroenterol. Hepatol. Bed. Bench. 2013; 6(1): 14–17. PubMed Abstract\n\nLiou K, Negishi K, Ho S, et al.: Detection of Obstructive Coronary Artery Disease Using Peak Systolic Global Longitudinal Strain Derived by Two-Dimensional Speckle-Tracking: A Systematic Review and Meta-Analysis. J. Am. Soc. Echocardiogr. Off. Publ. Am. Soc. Echocardiogr. 2016; 29(8): 724–735.e4. PubMed Abstract | Publisher Full Text\n\nVarat MA, Adolph RJ, Fowler NO: Cardiovascular effects of anemia. Am. Heart J. 1972; 83(3): 415–426. Publisher Full Text\n\nLester LA, Sodt PC, Hutcheon N, et al.: Cardiac Abnormalities in children with sickle cell anemia. Chest. 1990; 98(5): 1169–1174. Publisher Full Text\n\nWestwood MA, Shah F, Anderson LJ, et al.: Myocardial tissue characterization and the role of chronic anemia in sickle cell cardiomyopathy. J. Magn. Reson. Imaging. 2007; 26(3): 564–568. PubMed Abstract | Publisher Full Text\n\nCastro O, Hoque M, Brown BD: Pulmonary hypertension in sickle cell disease: cardiac catheterization results and survival. Blood. 2003; 101(4): 1257–1261. Publisher Full Text\n\nMushemi-Blake S, Melikian N, Drasar E, et al.: Pulmonary haemodynamics in sickle cell disease are driven predominantly by a high-output state rather than elevated pulmonary vascular resistance: a prospective 3-dimensional echocardiography/doppler Study. PLoS One. 2015; 10(8): e0135472. PubMed Abstract | Publisher Full Text\n\nWood JC, Tyszka JM, Carson S, et al.: Myocardial iron loading in transfusion-dependent thalassemia and sickle cell disease. Blood. 2004; 103(5): 1934–1936. PubMed Abstract | Publisher Full Text\n\nPoludasu S, Ramkissoon K, Salciccioli L, et al.: Left ventricular systolic function in sickle cell anemia: a meta-analysis. J. Card. Fail. 2013; 19(5): 333–341. PubMed Abstract | Publisher Full Text\n\nLamers L, Ensing G, Pignatelli R, et al.: Evaluation of left ventricular systolic function in pediatric sickle cell anemia patients using the end-systolic wall stress-velocity of circumferential fiber shortening relationship. J. Am. Coll. Cardiol. 2006; 47(11): 2283–2288. PubMed Abstract | Publisher Full Text\n\nWali YA, Venugopalan P, Rivera E, et al.: Cardiovascular function in omani children with sickle cell anaemia. Ann. Trop. Paediatr. 2000; 20(3): 243–246. Publisher Full Text\n\nChacko P, Kraut EH, Zweier J, et al.: Myocardial infarction in sickle cell disease: use of translational imaging to diagnose an under-recognized problem. J. Cardiovasc. Transl. Res. 2013; 6(5): 752–761. Publisher Full Text\n\nAhmed S, Siddiqui AK, Sadiq A, et al.: Echocardiographic abnormalities in sickle cell disease. Am. J. Hematol. 2004; 76(3): 195–198. Publisher Full Text\n\nAmzulescu MS, De Craene M, Langet H, et al.: Myocardial strain imaging: review of general principles, validation, and sources of discrepancies. Eur. Heart J. 2019; 20(6): 605–619.\n\nAhmad H, Gayat E, Yodwut C, et al.: Evaluation of myocardial deformation in patients with sickle cell disease and preserved ejection fraction using three-dimensional speckle tracking echocardiography: myocardial deformation in sickle cell disease. Echocardiography. 2012; 29(8): 962–969. PubMed Abstract | Publisher Full Text\n\nMartin CR, Johnson CS, Cobb C, et al.: Myocardial infarction in sickle cell disease. J. Natl. Med. Assoc. 1996; 88(7): 428–432.\n\nDabirian M, Janbabaei G, Karami H, et al.: Cardiac structural and functional changes evaluated by transthoracic and tissue doppler echocardiography in adult patients with sickle cell disease. Acta Inform. Medica. 2017; 25(1): 9–13. PubMed Abstract | Publisher Full Text\n\nMorissens M, Besse-Hammer T, Azerad M-A, et al.: Evaluation of cardiac function in patients with sickle cell disease with left ventricular global longitudinal strain. J. Transl. Intern. Med. 2020; 8(1): 41–47. Publisher Full Text\n\nBraga JCMS, Assef JE, Waib PH, et al.: Altered left ventricular twist is associated with clinical severity in adults and adolescents with homozygous sickle cell anemia. J. Am. Soc. Echocardiogr. 2015; 28(6): 692–699. Publisher Full Text\n\nSachdev V, Machado RF, Shizukuda Y, et al.: Diastolic dysfunction is an independent risk factor for death in patients with sickle cell disease. J. Am. Coll. Cardiol. 2007; 49(4): 472–479. PubMed Abstract | Publisher Full Text\n\nSachdev V, Sidenko S, Wu MD, et al.: Skeletal and myocardial microvascular blood flow in hydroxycarbamide-treated patients with sickle cell disease. Br. J. Haematol. 2017; 179(4): 648–656. Publisher Full Text\n\nGeard A, Pule GD, Chelo D, et al.: Genetics of sickle cell-associated cardiovascular disease: an expert review with lessons learned in Africa. Int. J. Integr. Biol. 2016; 20(10): 581–592.\n\nMunung NS, Nembaware V, de Vries J , et al.: Establishing a multi-country sickle cell disease registry in Africa: ethical considerations. Front. Genet. 2019; 10: 943. PubMed Abstract | Publisher Full Text\n\nGlassberg JA, Linton EA, Burson K, et al.: Publication of data collection forms from NHLBI funded sickle cell disease implementation consortium (SCDIC) registry. Orphanet J. Rare Dis. 2020; 15(1): 178. PubMed Abstract | Publisher Full Text\n\nZnegui T:ECHOCARDIOGRAPHIC EVALUATION IN CHILDREN WITH SICKLE CELL ANEMIA: CONTRIBUTION OF 2D STRAIN. figshare. [Dataset].2022. Publisher Full Text"
}
|
[
{
"id": "154031",
"date": "31 Oct 2022",
"name": "Hassen Ibn Hadj Amor",
"expertise": [
"Reviewer Expertise cardiology",
"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\nSickle cell disease is endemic in north Africa. It used to be a life-threatening disease with reduced life expectancy which improved in the recent years due to healthier lifestyles, medical monitoring, and new therapies. Nevertheless, we attest to an increase of chronic multi-systemic complications in addition to acute complications, mainly cardiovascular ones. The authors highlighted in this paper the importance of early detection of myocardial damage using echocardiographic techniques such as two-dimensional (2D) strain during childhood. It is a patient control study with a predictive factor analysis for alteration of the global longitudinal strain of the left ventricle. Generally, the manuscript is well written and the topic is quite interesting for the reader. The paper seems suitable for indexing in its actual version.\n\nIs the work 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": "154030",
"date": "03 Nov 2022",
"name": "Anthony Matta",
"expertise": [
"Reviewer Expertise Cardiovascular 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\nI have reviewed the submitted paper entitled “Evaluation of left ventricular systolic function in children with sickle cell anemia: contribution of 2D strain”. The authors showed that left ventricular global longitudinal strain is significantly altered in the SCD-group. They also suggested that treatment with hydroxycarbamide is a predictive factor.\nThis paper is not well written and not suitable for indexing in the current form. Major revision is required before being considered for further evaluation. Herein, I addressed the main points:\nEnglish editing is needed through all the manuscript.\n\nThe authors must define each abbreviation before its first use in the text (C means control in the abstract section).\n\nConclusion in abstract section is out of nowhere.\n\nIn the introduction section, the authors should cite the potential cardiac complications of sickle cell disease and explain in detail the two-dimensional strain technique.\n\nMethods section. It is completely unclear how the authors have performed this study. First, it is a case control study. Inclusion and exclusion criteria are not well defined. They mentioned among inclusion criteria: “having SCA and a performed TTE”, then they mentioned in the text that they performed TTE for all patients. So why do they fix the performance of TTE during follow-up as an inclusion criterion? When have they performed TTE? Did they compare the performed TTE to the previous ones?\n\nWhat was the indication for TTE in SCA or controls group? What was the indication for hospital admission in controls group?\n\nA comparative table with 4 columns should be added in the result section (Variable, SCA-group, C-group, p-value). This table should figure baseline characteristics (age, sex, BMI, laboratory values (Hb), treatment, dose, mean of LVGLS, LVEF, etc.).\n\nHow much was the proportion of patients with impaired LVGLS? Also, a clear definition of impaired LVGLS is lacking.\n\nMultivariable logistic regression investigating the association between impaired LVGLS and other parameters (Hydroxycarbamide dose, osteonecrosis, splenomegaly) was not performed. Then, a conclusion only based on bivariate analysis is extremely weak.\n\nI suggest putting 3 main tables in the results section: Table 1 (point 6), Table 2 (Impaired LVGLS vs normal LVGLS), Table 3 (multivariable logistic regression).\n\nWas a single TTE performed to each study participant? Or were repeated TTE performed?\n\nClear definitions of left ventricular dilation and hypertrophy were missed. All these terms must be defined in the method section including for threshold of normal LVGLS\n\nThe authors should mention in the result section that LVGLS was normal in all study participants in C-group.\n\nIt is true that there was a significant difference in echocardiographic parameters between study groups, but the represented values are within range of normal in both groups. They were not suitable for neither hypertrophic nor dilated cardiomyopathy diagnosis.\n\nThe authors should represent their results in the first paragraph of the discussion section. Then, they must focus on discussing their findings based on literature data.\n\nThey claim that hydroxycarbamide treatment is the predictor of LVGLS impairment, but in fact, the hydroxycarbamide dosage differs significantly between study groups. In the concerned table, the authors differ hydroxycarbamide treatment from hydroxycarbamide dose.\n\nThe authors should explain what this study adds to what is already known in the field. Previous studies investigate the role of LVGLS to predict cardiac outcomes in SCD patients.\n\nRe-write the conclusion (rephrase the first sentence which must highlight your take-home message).\n\nIt is unclear if a LVGLS below -20% is a predictor for cardiac disease in the future. It could be an incidental finding without clinical relevance. Usually, A GLS higher than 18% is considered normal.\n\nIs the work clearly and accurately presented and does it cite the current literature? No\n\nIs the study design appropriate and is the work technically sound? Partly\n\nAre sufficient details of methods and analysis provided to allow replication by others? Partly\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\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": "9051",
"date": "05 Dec 2022",
"name": "Tasnim Znegui",
"role": "Author Response",
"response": "1. English editing is needed through all the manuscript. We revised the manuscript. 2. The authors must define each abbreviation before its first use in the text (C means control in the abstract section). We rectified all abbreviations in the text. 3. Conclusion in abstract section is out of nowhere. We corrected the conclusion in the abstract section. 4. In the introduction section, the authors should cite the potential cardiac complications of sickle cell disease and explain in detail the two-dimensional strain technique. We added a paragraph to explain this. 5. Methods section. It is completely unclear how the authors have performed this study. First, it is a case control study. Inclusion and exclusion criteria are not well defined. They mentioned among inclusion criteria: “having SCA and a performed TTE”, then they mentioned in the text that they performed TTE for all patients. So why do they fix the performance of TTE during follow-up as an inclusion criterion? When have they performed TTE? Did they compare the performed TTE to the previous ones? It’s a cross sectional case-control study The (SCA) group included children followed up at the pediatric department of the Tunis military hospital for sickle cell disease during the study period. We did not include patients with SCA with history of heart diseases, or those who had undergone cardiac surgery. The control (C) group selected 30 children with no history of cardiovascular or respiratory pathology and not presenting anemia at the time of the study. Then, C group was matched to the SCA group by age, sex and body mass index. 6. What was the indication for TTE in SCA or controls group? What was the indication for hospital admission in controls group? These children had been hospitalized in the pediatric department and referred to a pediatric cardiology consultation for assessment of a heart murmur or for exploration of atypical chest pain, and who had a normal echocardiography. 7. A comparative table with 4 columns should be added in the result section (Variable, SCA-group, C-group, p-value). This table should figure baseline characteristics (age, sex, BMI, laboratory values (Hb), treatment, dose, mean of LVGLS, LVEF, etc.). We added a table untitled ‘Comparison of baseline characteristics of the two groups’. 8. How much was the proportion of patients with impaired LVGLS? Also, a clear definition of impaired LVGLS is lacking. In SCA group, LVGLS was estimated at -21.2±3%. It was significantly decreased (< -20%) in 46% of children (n=14) referring to ‘Reference Ranges of Left Ventricular Strain Measures by Two-Dimensional Speckle-Tracking Echocardiography in Children A Systematic Review and Meta-Analysis. J Am Soc Echocardiogr. 2016 Mar;29(3):209-225.e6. Epub 2015 Dec 30. 9. Multivariable logistic regression investigating the association between impaired LVGLS and other parameters (Hydroxycarbamide dose, osteonecrosis, splenomegaly) was not performed. Then, a conclusion only based on bivariate analysis is extremely weak. In multivariate analysis we found a correlation between LVM and impaired GLS (b = - 0.082, p<0.001) 10. I suggest putting 3 main tables in the results section: Table 1 (point 6), Table 2 (Impaired LVGLS vs normal LVGLS), Table 3 (multivariable logistic regression). We have organized the results section. 11. was a single TTE performed to each study participant? Or were repeated TTE performed? A single TTE was performed to each study participant. 12. Clear definitions of left ventricular dilation and hypertrophy were missed. All these terms must be defined in the method section including for threshold of normal LVGLS We revised the methodology. 13. The authors should mention in the result section that LVGLS was normal in all study participants in C-group. We mentioned this result. 14. It is true that there was a significant difference in echocardiographic parameters between study groups, but the represented values are within range of normal in both groups. They were not suitable for neither hypertrophic nor dilated cardiomyopathy diagnosis. We added a Table to compare the LV morphological parameters and LV systolic function in the two groups. 15. the authors should represent their results in the first paragraph of the discussion section. Then, they must focus on discussing their findings based on literature data. We corrected the first paragraph of the discussion section. 16. they claim that hydroxycarbamide treatment is the predictor of LVGLS impairment, but in fact, the hydroxycarbamide dosage differs significantly between study groups. In the concerned table, the authors differ hydroxycarbamide treatment from hydroxycarbamide dose. We corrected the correlation between treatment and LVGLS impairment. 17. the authors should explain what this study adds to what is already known in the field. Previous studies investigate the role of LVGLS to predict cardiac outcomes in SCD patients. The aim of this study was to assess the contribution of 2D strain to the detection of subclinical LV myocardial damage in children with SCA and to reveal associated factors with abnormal LVGLS. 18. Re-write the conclusion (rephrase the first sentence which must highlight your take-home message). We have corrected the conclusion."
},
{
"c_id": "9054",
"date": "05 Dec 2022",
"name": "Tasnim Znegui",
"role": "Author Response",
"response": "We are grateful to the reviewers for their insightful comments."
}
]
}
] | 1
|
https://f1000research.com/articles/11-1207
|
https://f1000research.com/articles/11-1430/v1
|
05 Dec 22
|
{
"type": "Research Article",
"title": "Enhancement of surface properties of polyetheretherketone implant material by fractional laser texturing",
"authors": [
"Mustafa S. Tukmachi",
"Hikmat J. Abdul-Baqi",
"Falah H. Hussein",
"Hikmat J. Abdul-Baqi",
"Falah H. Hussein"
],
"abstract": "Background: Polyetheretherketone (PEEK) is a promising implant material due to its superior biomechanical strength. However, due to its hydrophobic nature and lack of cellular adhesion properties, it has poor integration with bone tissue. Methods: A fractional CO2 laser was used with various parameters for surface texturing of PEEK substrate to enhance its surface properties. An optical microscope and field-emission scanning electron microscope (FESEM) were used to examine the surface morphology of untextured and laser-textured samples. Energy dispersive X-ray spectroscopy (EDX) was performed to determine the effect of the laser on the microstructure of PEEK. Surface microroughness, atomic force microscopy (AFM), and wettability were investigated. Results: There were significant increases in microroughness, nanoroughness, surface area ratio, and wettability after laser texturing with no change in the elemental composition. The best results were obtained by using 400 µs laser pulse duration with a dot separation distance of 0.2 mm and a 60° staggered dots pattern. Conclusions: Laser surface texturing of PEEK implant material by fractional CO2 laser is an easy and fast method of introducing patterned topographical features with no need for additional devices. With further investigations, this method of PEEK modification might have the potential to be used in the implant field.",
"keywords": [
"PEEK",
"laser surface texturing",
"fractional CO2 laser",
"roughness",
"wettability"
],
"content": "Introduction\n\nPolyetheretherketone (PEEK) is a high-performance polymer that has been recently investigated as an alternative biomaterial for metallic materials in dentistry.1 The increased interest in PEEK as an implant material is due to its biocompatibility and its superior physical and mechanical properties over titanium and other metallic materials.2,3\n\nStudies have shown that PEEK causes fewer hypersensitive and allergic reactions than titanium.4,5 Radiolucency is one of the benefits of using PEEK as an implant material since it can be imaged by X-ray without any distortion or artifacts.6,7 Besides, it has more aesthetic appeal than titanium due to its beige color.8 Most importantly, the elastic modulus value of neat PEEK is about 3.6 GPa, which lies between the values of trabecular (1.4 GPa) and cortical bone (14 GPa).9,10 The close proximity in stiffness between PEEK and human bone causes less stress shielding and bone resorption around the implant in comparison with stiffer titanium (elastic modulus = 110 GPa).11\n\nThis polymer is not bioactive due to its hydrophobicity and poor interfacial interaction, meaning it has a limited ability to bind with the surrounding bone tissue. Wettability is a crucial property of an implant material since the first event of implantation is the adsorption of water and protein from the blood on the implant surface, which signals the process of new bone formation.12,13 However, it is well-established that wettability is affected by the biomaterials' surface roughness and texture, which play important roles in establishing a functional interface with the surrounding tissue in the body.14,15\n\nThe topography of a surface has a direct impact on biological reactions at the cellular level, including cell orientation and migration as well as the development of ordered cytoskeletal structures. Successful osseointegration of implants has been shown to be associated with surface roughness in the nano- and micro-scales.16,17 Previous studies18,19 demonstrated that a moderately rough implant surface with an average surface roughness of more than 1 μm allowed for bone ingrowth and provided mechanical interlocking with bone tissue. High bone-to-implant contact was seen with rough surfaces due to the increase in surface area.18,19\n\nSeveral methods have been experimented to alter the surface topography of PEEK polymer, including acid etching, grit blasting, plasma treatment, and laser texturing.20 Laser surface texturing (LST) is one of the simplest methods to modify the surface of polymers. It can simultaneously alter the surface roughness at the macro-, micro-, and nano-sized scales without using different tools.21 A laser can be used to create surface features like pits and grooves with an equidistance ranging from several nanometers to even micrometers between the pits or grooves.22 These topographic features influence cellular adhesion, migration, proliferation, and differentiation behavior.23,24\n\nA fractionated laser is a mode of laser delivery in which energy is conveyed in an array of parallel vertical columns of multiple microscopical thermal spots termed microscopic treatment zones (MTZs) with a constant distance between spots.25 Fractional CO2 was introduced in 2004 in dermatology for skin resurfacing and it may have several advantages in dentistry, especially in the surface treatment of biomaterials.26–28 The device allows for a precise irradiation area with a more homogenous texturing pattern to be set without the need for manual movement of the device handpiece. Unlike with traditional CO2 lasers, there is less overall material bulk heating and thermal degradation with fractional lasers.25 Additionally, with the correct selection of laser parameters (power, pulse duration, distance between spots, and spot pattern), the desired melting, ablation, and penetration depth can be achieved.29,30\n\nThe aim of this research was to improve the surface roughness and wettability of a PEEK substrate by fractional CO2 laser texturing without affecting the material’s microstructure. The null hypothesis is that laser surface texturing by fractional CO2 laser does not improve the surface roughness nor the wettability of PEEK.\n\n\nMethods\n\nPEEK samples were supplied by (Energetic Industry Co., Ltd., China) by means of cutting continuous extruded rods into discs with dimensions of 10 mm × 2 mm (diameter × thickness). The samples were gradually ground with 500, 800, 1200, 2000, and 2400 grit silicon carbide abrasive papers, and they were polished to obtain a mirror finish. After that, all samples were cleaned with ethanol and water, successively.\n\nLaser surface texturing of PEEK samples was carried out using a fractional CO2 laser system (CICU-f, Ilooda Co., Ltd., South Korea). This device has a fixed output power of 11.25 W and a laser wavelength of 10600 nm. Different pulse durations (PD) were tried (100, 200, 300, 400, and 500 μs) and two patterns (straight dots and 60° staggered dots) were performed for each pulse duration at a dot separation distance of 0.2 mm. The handpiece of the device was affixed by a clamp to prevent any movement during the procedure and to ensure the incident laser beam was perpendicular to the sample (0° incidence angle). The texturing procedure was done with 1 scan (pass). After laser treatment, the samples were cleaned with ethanol in an ultrasonic cleaning device.\n\nInitial examination of laser texturing was done using an optical microscope (Olympus BH-2, Japan) at 10× magnification. The observations included the effect of laser heating on PEEK material and any signs of burning or carbonization. The samples that were treated with 100 μs PD did not show any observable laser effect on the PEEK surface, and those treated with 500 μs PD resulted in the burning of the material. For those reasons, these parameters were not considered for further surface characterizations.\n\nThe surface morphology of untreated (control) and laser treated PEEK samples was examined by field emission scanning electron microscopy (FESEM) (MIRA3, TESCAN, Czech Republic) to observe the texturing pattern, spot shape, melting pattern around the spot, ablation at the heat-affected zone, and presence of cracks. Energy dispersive X-ray spectroscopy (EDX) was performed to determine the effect of laser on the microstructure of the material. Elemental weight percentages and atomic percentages were compared between control samples and laser treated samples. For laser treated samples, elemental analysis was done in three areas: at the center of the dot, at the edge of the dot, and outside the dot (within the heat-affected zone).\n\nSurface microroughness was measured by a digital profilometer (TR220, Beijing Time High Technology Ltd., China). The device has a sharp stylus with an 8 mm travelling distance across the surface. Three readings of the average surface roughness (Ra) in micrometers were obtained for each sample, and the average of these readings was recorded for each sample.\n\nA contacting AFM device (BenYuan CSPM-5500, Being Nano-Instruments Ltd., China) was used to obtain 3D topographical images of PEEK samples. The AFM was operated in tapping mode with a 299.6 kHz tapping frequency. The scanning area was a square with a size of about 4550 × 4550 nm. The average surface roughness (Sa) and the surface area ratio (Sdr) were obtained for each sample.\n\nThe wettability of PEEK samples was assessed by a contact angle goniometer (Cam110 contact angle goniometer, Creating Nano Technologies Inc., Taiwan) with a sessile drop of deionized water. The device consists of a table for holding the sample, a syringe with a revolving knob on top to release the water drop, and a camera that is connected to a computer to analyze the image with an included imaging software (Touchmate, Cam110). The sample was placed on the table and a 7 μm drop of deionized water was released from the syringe. The water drop was left to spread on the sample for 30 seconds, and then an image was captured. The computer software was used to calculate the contact angles.\n\nPrism 8 (GraphPad Software, USA) (RRID:SCR_002798) was used for statistical analysis of the data (an open-source alternative able to perform similar analysis is JASP). The results were depicted as bar charts, with the mean values (n = 10) placed inside the bars and the standard deviation written above the bars. To assess for statistical significance among groups, one-way analysis of variance (ANOVA) was used, and for multiple comparisons, Tukey's HSD (honestly significant difference) post-hoc test was performed. A P-value > 0.05 is statistically non-significant (NS), < 0.05 is significant (S), and < 0.01 is highly significant (HS).\n\n\nResults\n\nOptical microscope images of control and laser textured samples36 are shown in Figure 1. Samples treated with 100 μs PD did not show any noticeable laser effect for both patterns. With increasing the pulse duration over 200 μs, the dots pattern became more prominent with larger heat-affected zones. The dots of samples with 400 μs PD had more depth than other samples with less PD due to the melting and evaporation of the material as the absorbed heat increased. Carbonization can be seen over the edges of the dots with samples treated with 500 μs PD. The staggered dots pattern seemed to cover more surface area than the straight dots pattern with fewer unaffected zones between the dots due to dots staggering or zig-zag dots formation.\n\nThe surface morphologies of PEEK samples at 200× and 2000× magnifications are shown in Figure 2 and Figure 3, respectively. The FESEM images were consistent with the optical microscope images in terms of the effect of heat on the material. The laser effect became more prominent as the pulse duration was increased. With PD of 200 μs, there was some uniform ablation at the center of the dot and in the heat-affected zone with no signs of melting. There was little melting at the center of the dot with 300 μs PD, but with 400 μs PD complete melting occurred at the center of the dot with formation of uniform edges. There were no cracks caused by laser in all samples.\n\nElemental analysis with an EDX spectrum of untreated PEEK sample is presented in Figure 4. An area analysis was done for the whole image shown in the figure. For laser treated sample with 400 μs PD, EDX spectra were obtained at three points, as shown in Figure 5 to assess the effect of laser on the microstructure of the material. The elemental analysis of the laser treated samples showed very comparable weight percentages of carbon and oxygen to that of unmodified samples. There was no increase in the percentage of carbon at the center or at the margins of the spot, indicating no presence of carbonization or burning.\n\nMean values of surface roughness (Ra) in micrometers are presented in Figure 6. One-way ANOVA showed that there was a high significance among groups (p-value < 0.01). All laser treated samples showed a highly significant increase in surface roughness compared to the control sample. The highest microroughness values belonged to samples treated with 400 μs pulse duration and a staggered dots pattern.\n\nThe 3-dimensional topographical images of control and laser textured samples are presented in Figure 7. There was an increase in summits and valleys density in all laser treated samples compared to the control group.\n\nMean values of AFM average surface roughness (Sa) and surface area ratio (Sdr) are presented in Figure 8 and Figure 9, respectively. There were highly significant increases in Sa and Sdr values for all laser samples in comparison to control samples. The samples of 400 μs PD treatment with a staggered dots pattern showed the highest mean values of both Sa and Sdr.\n\nImages of water contact angle measurements are shown in Figure 10, while the mean values are presented in Figure 11. One-way ANOVA resulted in a high significance among groups. All laser textured samples showed a highly significant decrease in the water contact angle. There were highly significant improvements in wettability with laser PD of 400 μs especially with a staggered dots pattern, which showed the most improvement in this property.\n\n\nDiscussion\n\nThis study was conducted to assess the efficiency of a fractional CO2 laser system used for skin resurfacing in surface texturing and enhancement of surface properties of PEEK implant substrate. PEEK has proven itself to be a valuable material in the field of implantology due to its biocompatibility, high mechanical strength, inherent radiolucency, and exceptional chemical resistance. Unfortunately, PEEK's bioinert surface does not support cellular adhesion and bone tissue bonding, limiting its use in the medical field.12\n\nLaser texturing with a fractional laser is easy, fast, and applicable since the whole surface can be treated with a single run without the need for a moving device. When a laser beam of high intensity comes in contact with the surface of a material, some of the beam is absorbed by the material, and the remaining intensity is lost to the surrounding environment through the processes of reflection and scattering. The effect of laser on a polymeric material can be either photothermal or photochemical. A photothermal effect occurs when the absorbed laser energy converts to heat that raises the temperature of the material. When the temperature of the substance exceeds its boiling point, it turns into vapor and is generally ejected from the surface as it boils. This effect induces diverse phenomena, including melting or vaporization (ablation), which modify the topography.31\n\nThe photochemical effect occurs when the laser used has a wavelength in the UV range (like excimer lasers) in which the photon energy is enough to break molecular bonds at the polymer surface and induce a change in the surface chemistry.\n\nSince the wavelength of CO2 laser is within the infrared range (10600 nm), the effect induced on the PEEK surface was photothermal by a process called photothermolysis without any chemical change in the microstructure of the material as confirmed by EDX in Figure 4 and Figure 5. The fractional laser causes selective photothermolysis by splitting the laser beam into an array of microbeams with a fixed distance between them. The ablation and texturing pattern of PEEK material became clearer and more distinct as the pulse duration was increased, as can be seen in Figure 1. By increasing the pulse duration, the spot energy increases and causes more heat to be absorbed by the material. With a short pulse duration of 100 μs, no effect was seen because the heat was below the melting threshold of PEEK, while increasing the duration to 500 μs caused burning of the material due to intense heating.\n\nSurface topography, roughness, and wettability have been shown to significantly affect cellular responses to biomaterials in a number of studies.32–34 The behavior of cells on biomaterial surfaces is determined by the interaction between implant and cells, which is related to surface roughness. Naturally, bone cells encounter and interact with nanostructures in their environment, such as extracellular matrix (ECM) proteins. To improve bone cell adhesion and proliferation, it is thoughtful to mimic the cellular environment by creating materials with nanotopography. While nanotopographic features are important for the recruitment and migration of bone cells to the implant surface, microtopographically complex surfaces are equally vital in increasing the degree of bone-to-implant contact. Microcraters created on PEEK’s surface by the fractional laser could encourage bone ingrowth and enhance the anchorage of implants with bone tissue. The results of this study confirmed that laser texturing with fractional CO2 laser significantly increase surface roughness on both nano- and micro-level as demonstrated by AFM and microroughness tests. The samples treated with 400 μs pulse duration showed surface roughness values of about 1.3 μm. This value is well within the optimum roughness range (1–2 μm) to enhance bone integration reported by other studies.32,35\n\nIn regards to wettability, it is an essential property for cell spreading, which in turn is an important stage in cell adhesion prior to proliferation and differentiation. The wettability was significantly enhanced after laser texturing, particularly with samples treated with 400 μs PD which showed the least water contact angles among other samples. These results are consistent with Wenzel’s theory, which stated that adding surface roughness and increasing the surface area leads to an enhancement in the wettability.\n\nSince the fractional CO2 laser surface texturing improved the surface properties of PEEK material, the null hypothesis was rejected.\n\n\nConclusion\n\nAlthough this study has some limitations, it can be concluded that laser texturing of PEEK biomaterial by fractional CO2 laser significantly enhanced its surface properties in terms of surface topography, roughness, and wettability. The best results were achieved with pulse duration = 400 μs, dot separation distance = 0.2 mm, and a 60° staggered dots pattern. With further in vitro (like cell culture) and in vivo (like animal study) investigations, this method of PEEK modification might have the potential to be used in the implant field.",
"appendix": "Data availability\n\nFigshare: Enhancement of surface properties of polyetheretherketone implant material by fractional laser texturing. https://doi.org/10.6084/m9.figshare.21518136.v5. 36\n\nThis project contains the following underlying data:\n\n- Surface microroughness.csv (Raw data of surface microroughness test)\n\n- AFM Sa (nm).csv (Raw data of AFM average surface roughness)\n\n- AFM Sdr.csv (Raw data of AFM surface area ratio)\n\n- Wettability.csv (Raw data of water contact angle measurements)\n\n- Microscope images.rar (All raw images taken with microscopes)\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).\n\n\nReferences\n\nKadhum AS, Alhuwaizi AF: The effect of composite bonding spot size and location on the performance of poly-ether-ether-ketone (PEEK) retainer wires. J. Bagh. Coll. Dent 2021; 33(2): 1–9. Publisher Full Text\n\nKurtz SM: An Overview of PEEK Biomaterials, in PEEK Biomaterials Handbook. Kurtz SM, editor. Oxford:William Andrew Publishing;2012; p. 1–7.\n\nSkirbutis G, Dzingutė A, Masiliūnaitė V, et al.: A review of PEEK polymer's properties and its use in prosthodontics. Stomatologija Baltic Dental and Maxillofacial Journal. 2017; 19(1): 19–23. PubMed Abstract\n\nGoutam M, Giriyapura C, Mishra SK, et al.: Titanium allergy: a literature review. Indian J. Dermatol. 2014; 59(6): 630–634. PubMed Abstract | Publisher Full Text\n\nKofler L, Wambacher M, Schweinzer K, et al.: Allergic Reaction to Polyether Ether Ketone Following Cross-Reactivity to Epoxy Resin. J. Cutan. Med. Surg. 2017; 21(1): 78–79. PubMed Abstract | Publisher Full Text\n\nGupta S, Patil N, Solanki J, et al.: Oral Implant Imaging: A Review. Malays. J. Med. Sci. 2015; 22(3): 7–17.\n\nLaux CJ, Hodel SM, Farshad M, et al.: Carbon fibre/polyether ether ketone (CF/PEEK) implants in orthopaedic oncology. World J. 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Publisher Full Text\n\nNieminen T, Kallela I, Wuolijoki E, et al.: Amorphous and crystalline polyetheretherketone: Mechanical properties and tissue reactions during a 3-year follow-up. J. Biomed. Mater. Res. A. 2008; 84(2): 377–383. PubMed Abstract\n\nModaresifar K, Hadjizadeh A, Niknejad H: Design and fabrication of GelMA/chitosan nanoparticles composite hydrogel for angiogenic growth factor delivery. Artif. Cells Nanomed. Biotechnol. 2018; 46(8): 1799–1808. PubMed Abstract | Publisher Full Text\n\nHassan AH, Al-Judy HJ: Biomechanical Effect of Nitrogen Plasma Treatment of Polyetheretherketone Dental Implant in Comparison to Commercially Pure Titanium. J. Res. Med. Dent. Sci. 2018; 6(2): 367–377.\n\nRupp F, Scheideler L, Rehbein D, et al.: Roughness induced dynamic changes of wettability of acid etched titanium implant modifications. Biomaterials. 2004; 25(7-8): 1429–1438. PubMed Abstract | Publisher Full Text\n\nMartínez E, Engel E, Planell JA, et al.: Effects of artificial micro- and nano-structured surfaces on cell behaviour. Annals of Anatomy - Anatomischer Anzeiger. 2009; 191(1): 126–135. PubMed Abstract | Publisher Full Text\n\nSimon Z, Watson PA: Biomimetic dental implants--new ways to enhance osseointegration. Journal of the Canadian Dental Association. Journal de L'Association Dentaire Canadienne. 2002; 68(5): 286–288.\n\nAlbrektsson T, Wennerberg A: On osseointegration in relation to implant surfaces. Clin. Implant. Dent. Relat. Res. 2019; 21 Suppl 1: 4–7. PubMed Abstract | Publisher Full Text\n\nKnaus J, Schaffarczyk D, Cölfen H: On the Future Design of Bio-Inspired Polyetheretherketone Dental Implants. Macromol. Biosci. 2020; 20(1): 1900213–1900239. (1900239). Publisher Full Text\n\nRiveiro A, Maçon ALB, del Val J , et al.: Laser Surface Texturing of Polymers for Biomedical Applications. Front. Phys. 2018; 6(16): 1–17.\n\nAl-Khafaji AM, Hamad TI: Assessment of Surface Roughness and Surface Wettability of Laser Structuring Commercial Pure Titanium. J. Res. Med. Dent. Sci. 2020; 8(1): 81–85.\n\nBettinger CJ, Langer R, Borenstein JT: Engineering substrate topography at the micro- and nanoscale to control cell function. Angewandte Chemie. International Ed. In English. 2009; 48(30): 5406–5415. PubMed Abstract | Publisher Full Text\n\nRiveiro A, Soto R, Comesaña R, et al.: Laser surface modification of PEEK. Appl. Surf. Sci. 2012; 258(23): 9437–9442. Publisher Full Text\n\nAhrari F, Heravi F, Hosseini M: CO2 laser conditioning of porcelain surfaces for bonding metal orthodontic brackets. Lasers Med. Sci. 2013; 28(4): 1091–1097. PubMed Abstract | Publisher Full Text\n\nManstein D, Herron GS, Sink RK, et al.: Fractional Photothermolysis: A New Concept for Cutaneous Remodeling Using Microscopic Patterns of Thermal Injury. Lasers Surg. Med. 2004; 34(5): 426–438. PubMed Abstract | Publisher Full Text\n\nKhan MH, Sink RK, Manstein D, et al.: Intradermally focused infrared laser pulses: Thermal effects at defined tissue depths. Lasers Surg. Med. 2005; 36(4): 270–280. PubMed Abstract | Publisher Full Text\n\nAyyash MH, Mahmood AS: Fractional CO2 Laser Treatment of Mild Periorbital Wrinkles in Iraqi Patients. Iraqi J. Laser. 2020; 18(2): 21–26.\n\nLawrence J, Li L: Wettability characteristics of carbon steel modified with CO2, Nd:YAG, excimer and high power diode lasers. Appl. Surf. Sci. 2000; 154-155: 664–669. Publisher Full Text\n\nMontealegre MA, Castro G, Rey P, et al.: SURFACE TREATMENTS BY LASER TECHNOLOGY. Contemp. Mater. 2010; 1: 19–30. Publisher Full Text\n\nTan WS, Zhou JZ, Huang S, et al.: Fabrication of polymer microcomponents using CO2 laser melting technique. POLIMERY. 2015; 60(3): 192–198. Publisher Full Text\n\nMartin JY, Schwartz Z, Hummert TW, et al.: Effect of titanium surface roughness on proliferation, differentiation, and protein synthesis of human osteoblast-like cells (MG63). J. Biomed. Mater. Res. 1995; 29(3): 389–401. PubMed Abstract | Publisher Full Text\n\nPonsonnet L, Reybier K, Jaffrezic N, et al.: Relationship between surface properties (roughness, wettability) of titanium and titanium alloys and cell behaviour. Mater. Sci. Eng. C. 2003; 23(4): 551–560. Publisher Full Text\n\nHao L, Lawrence J, Chian KS: Osteoblast cell adhesion on a laser modified zirconia based bioceramic. J. Mater. Sci. Mater. Med. 2005; 16(8): 719–726. PubMed Abstract | Publisher Full Text\n\nRosales-Leal JI, Rodríguez-Valverde MA, Mazzaglia G, et al.: Effect of roughness, wettability and morphology of engineered titanium surfaces on osteoblast-like cell adhesion. Colloids Surf. A Physicochem. Eng. Asp. 2010; 365(1): 222–229. Publisher Full Text\n\nTukmachi M, Abdul-Baqi H, Hussein F:Enhancement of surface properties of polyetheretherketone implant material by fractional laser texturing. figshare. [Dataset].2022. Publisher Full Text"
}
|
[
{
"id": "157476",
"date": "19 Dec 2022",
"name": "Kadhim A. Hubeatir",
"expertise": [
"Reviewer Expertise Laser Material interactions applications in medical and scientific 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\n1. You must explain the null hypothesis as compared with this research in the introduction.\n2. In Methods:\nMy question is, are the dimensions of the prepared samples standard?\n3. In results:\nOptical light microscope don't need reference especially for Fig (1).\n4. In Discussion:\nIn page 11 at the end of this page you must write the amount percentage of absorption of PEEK to wavelength 10600 nm (CO2 Laser).\n\nIn Page 12 below Fig. 11, line 3 needs a references or evidence to confirm this explanation: \"The fractional laser causes selective photothermolysis by splitting the laser beam into array of micro beams with a fixed distance between them\".\n5. References:\nAll of them are checked it was new and good writing.\n\nIs the work 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": "257219",
"date": "04 Apr 2024",
"name": "Rajeev Verma",
"expertise": [
"Reviewer Expertise Surface Egnieering"
],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThere is enough scientific content in the article, however, the article hardly contains any discussion on the results obtained in the study. Review Comments:\nRegarding LST various parameters can affect the surface such as frequency, and scanning speed so authors must mention these values also. In the discussion section, the last line is incomplete or not related to context “Since the fractional CO2 laser surface texturing improved the surface properties of PEEK material, the null hypothesis was rejected.” Wettability can be varied with the size of the spot also so, it's better to mention the diameter of the spot and pitch values. The discussion section looks more like the introduction section; the authors should discuss the results more clearly. In Fig 2 and Fig 3 at 200 μs staggered dot is brighter than the straight dot at the same laser parameters, what could be the reason the authors should provide some justification for that? In Fig 2 and Fig 1 spot at 200 μs looks bigger than 300 and 400 μs, which could be the reason authors should provide some justification for that. In fig 6 and 8 average surface roughness values are presented but, there is a huge difference between both values. So, which value should be considered and why? The authors should discuss these.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nNot applicable\n\nAre all the source data underlying the results available to ensure full reproducibility? No\n\nAre the conclusions drawn adequately supported by the results? Partly",
"responses": []
}
] | 1
|
https://f1000research.com/articles/11-1430
|
https://f1000research.com/articles/11-694/v1
|
23 Jun 22
|
{
"type": "Research Article",
"title": "Current views of community and hospital pharmacists on pharmaceutical care services in the United Arab Emirates: A mixed methodological study",
"authors": [
"Zelal Kharaba",
"Joviana Farhat",
"Bassam S. Mahboub",
"Manal Ali Buabeid",
"Yassen Alfoteih",
"Yaser Al-Worafi",
"Ammar Jaber",
"Mohammad AlAhmad",
"Joviana Farhat",
"Bassam S. Mahboub",
"Manal Ali Buabeid",
"Yassen Alfoteih",
"Yaser Al-Worafi",
"Ammar Jaber"
],
"abstract": "Background: The profession of pharmacy has evolved significantly in recent years in terms of professional service delivery. The aim of this study was to explore the current views of pharmacists in the United Arab Emirates (UAE) on pharmaceutical care services and the nature of barriers encountered in practice using qualitative and quantitative assessment methods. Methods: A cross-sectional study was conducted among hospital and community pharmacists (n = 305) between March and May 2021, using qualitative and quantitative assessment methods. In the qualitative phase, 15 interviews were conducted to explore five main criteria: patient information, inadequate patient counseling, prescribing errors prevention and identifying drug-related problems, lack of participation in health awareness programs, and barriers to pharmaceutical care implementation. In the quantitative phase, 305 consenting pharmacists completed a questionnaire on seven criteria: demographic profile, pharmacist-physician interaction, patient counseling assessment, patient reports of adverse drug events, pharmacist participation in health awareness programs, perceptions of reducing prescribing errors and identifying drug-related problems, and barriers to appropriate pharmaceutical care implementation. Results: The results of both the qualitative and quantitative phases of the study revealed that pharmacists' influence on practice in the UAE is limited due to many factors, mainly lack of time and patients' ignorance of the pharmacist's role in the medical field. The mean responses regarding pharmacists' approach to patient counseling and patients' knowledge of pharmacists' role in managing adverse drug reactions were 77.1% and 59.7%, respectively. Active participation in health awareness programs was 64.8%. The mean positive response of participants in reducing prescribing errors and recognizing drug-related problems was 9.2%. Pharmacists' age and number of years in practice were the most important factors influencing the pharmaceutical care services implementation. Conclusion: The study has shown the need to shed light on the proper implementation of pharmaceutical care while maintaining a trusting relationship with physicians.",
"keywords": [
"hospital pharmacists",
"community pharmacists",
"pharmaceutical care services",
"interprofessional education."
],
"content": "Introduction\n\nTraditionally, pharmacy was considered a profession overlapping the health and chemical sciences, ensuring the safe use of medicines. Over time, the role of the pharmacist shifted from a product-oriented practice to a more patient-centered practice, giving rise to the concept of pharmaceutical care. Pharmaceutical care is the direct, responsible provision of medication-related care aimed at achieving specific outcomes and improving a patient’s quality of life.1 Accordingly, the interaction between pharmacists, physicians, and other health care professionals has been influenced. Indeed, a close working relationship between pharmacists and physicians is critical to optimizing patient care. Thus, the study of pharmacy has changed globally over the past 30 years and aims to enhance the medical skills acquired during the professional career. Communication skills, experience and vigilance are the main internal factors that influence performance. On the other hand, environmental factors such as availability of restrooms are important external factors that improve the work of pharmacists.\n\nNowadays, pharmacists work in many different settings such as communities, hospitals, industrial environments and healthcare service institutions.2 Consequently, continuous improvement of pharmacists’ experience and knowledge is the most important asset to achieve the highest possible level of professional execution through standardized goals that allow detailed access to drug therapy, optimization of patient outcomes, minimization of redundancies and interruptions in health care delivery, and increase of collaboration and synergy between pharmacists, physicians, and other health care professionals.3\n\nThe above achievements have given pharmacy practice a social impact as basic and pre-licensure education provides pharmacists with broad knowledge and expertise on all aspects of preparation, distribution, action and use of drugs and medicines.4\n\nTherefore, a sufficiently stored scientific discipline of mind enabled them to be efficient self-learners. Although pharmacists are now able to fill their role in the community, they are still underestimated in the Middle East, especially in the United Arab Emirates (UAE), as their role as one of the main chief contributors in the treatment, consultation and follow-up of patients is somehow ignored. A recently published study investigated the impact of clinical and non-clinical risk factors on pharmacists’ performance and practice of pharmacy in public pharmacies in Abu Dhabi. This study reported a large gap in medication errors and computer system malfunctions, followed by theft incidents, and violent behavior that manifested in verbal, psychological, and physical assaults.5\n\nFrom a statistical perspective, the distribution of pharmacists may vary from city to city, reflecting the shortage and uneven distribution of health professionals. On the other hand, other critical obstacles also contribute to the limitation of pharmacy practice, namely: demographic factors such as the aging of the population and the geographical distribution of people, economic factors mainly related to the increased health care costs and the growing gap between wealthy and poor people, and sociological factors such as the use of traditional medicines even in severe cases.6\n\nIn developing countries, the role and influence of pharmacists in society is still unclear and underappreciated. In a 2014 study on the role of pharmacists in the United Arab Emirates, the authors noted a number of challenges and barriers to optimizing pharmacy services.7 These could be due to a lack of resources to compensate and retain pharmacists in community and hospital pharmacies, overlapping responsibilities of healthcare professionals, and a lack of interprofessional collaboration.\n\nIn practice, it is difficult to achieve ideal interprofessional collaboration in health care because it is difficult to demonstrate that all members of the health care team are equally responsible for and committed to achieving a particular clinical goal. It appears that physicians take the dominant role as clinical leaders in the hierarchical health care system, which may be related to the difficulties in creating a collaborative work environment when the pharmacist is considered a member of lesser importance and significance.8 The result of this non-collaborative environment is a lack of coordination, sharing of information, experiences, publications, and research between pharmacists and other health professionals, leading to inadequate teamwork that impacts patient health. In addition, universities lack autonomy, governance, and institutional performance.9\n\nIn addition, there is a lack of linkage between pharmaceutical implementation and national sustainable development plans. In several countries, the implementation of pharmaceutical care still faces a number of obstacles. Chief among these are lack of time, limited access to patients’ medication records, unsuitable premises, an insufficient number of competent pharmacists and the absence of standard practice strategies.10–12\n\nFor this reason, this study will shed light on the current situation of pharmacy practice in the UAE while providing healthcare professionals with a suitable future-oriented work plan to help this profession become more influential in society.\n\n\nMethods\n\nThis study was performed based on the (STROBE) Guidelines for Strengthening the Reporting of Observational Studies in Epidemiology for reporting cross-sectional observational studies and the Consolidated Criteria for Reporting Qualitative Studies (COREQ).\n\nThe study was conducted among hospital and community pharmacists across all emirates in the UAE between March and May 2021, using quantitative and qualitative assessment methods. In the quantitative phase, a paper-based questionnaire was used and distributed among different hospital and community pharmacists by the research team. In the qualitative phase, interviews were conducted among pharmacists who agreed and consented to enroll in the interview phase of the study.\n\nA total of 400 questionnaires were distributed to pharmacists. Of these, 305 pharmacists agreed to participate and completed the questionnaire (response rate 76.3%), 36 of whom were hospital pharmacists and 269 of whom were community pharmacists. Inclusion criteria required that participants were licensed pharmacists working in community or hospital pharmacies in the UAE for at least two years. Pharmacy technicians, non-licensed pharmacists, assistants or interns were excluded from this study.\n\nBased on the previous literature and Raosoft sampling calculator, 300 responses for the quantitative phase and 15 for the qualitative phase were considered representative for this study.\n\nThe study received the required ethical approval from the Research Ethics Committee (REC) of Al-Ain University (AAU-REC -B3, Feb 2021). Informed verbal consent was obtained prior to data collection for the anonymous use and sharing of participant data.\n\nThe questionnaire was designed after reviewing several literature reviews from previous studies. Accordingly, Murtaza G et al.’s (2015) questionnaire was used in this study.13 Murtaza G et al.’s (2015) questionnaire was designed after a comprehensive review of literature and tested for validity and reliability.13 We adopted this questionnaire and modified it slightly to suit the purpose and population of the present study. This questionnaire was completed individually by all participants. The research assistants were also available to answer or explain the participants’ questions and minimize the possibility of missing information.\n\nThe questionnaire consisted of seven criteria: 1) vital statistics of the population, 2) pharmacist-physician interactivity, 3) pharmacist’s role and involvement in patient counseling, 4) handling and reporting of adverse drug reactions in patients, 5) participation in health awareness programs, 6) pharmacist’s role in minimizing prescribing errors and identifying drug-related problems, 7) barriers to implementing pharmaceutical care in the UAE.\n\nA non-probability convenient sampling technique was used for data collection due to the low willingness of pharmacists to complete the questionnaire and cooperate in the study. Participants underwent two phases of the study: quantitative and qualitative.\n\nThe quantitative phase focused on data collection through the distribution of a questionnaire using a non-systematic random sampling technique in all major cities of the UAE: Abu Dhabi, Dubai, Sharjah, Ajman, Umm Al Quwain, Ras Al Khaimah and Fujairah. The administered questionnaire was a written survey with eligible pharmacists who agreed to participate in the study. A copy of the questionnaire can be found under Extended data. The questionnaires were distributed by four research assistants who received a lecture on the topic and training on how to complete the questionnaire, all of which were conducted by one of main researchers (ZK). The research staff randomly approached eligible pharmacists in different community and hospital pharmacies in all emirates of the UAE. Participants were first informed of the purpose of the study and the estimated time required to complete the questionnaire. They were then given the choice of completing the questionnaire themselves or being supervised by a member of the study staff. Pharmacists were also informed of the anonymity and confidentiality of the study.\n\nIn the qualitative phase, a pilot interview was conducted. Participants were randomly selected and interviewed using a snowball approach. Participants were selected from the pool of participants from the quantitative phase. Only participants who agreed to be interviewed were considered in this phase of the study. The researcher (ZK) made an appointment with the participants by telephone and explained the aims and rationale for this research before conducting the interviews. Interviews were conducted face-to-face in hospital or community pharmacies for 30 minutes alone with (ZK), who is a female pharmacist and holds a PhD in clinical pharmacy and Good Clinical Practice (GCP) certificate. The saturation point was reached after 15 field note interviews. Field notes were completed during the interviews and discussed with the participants before they were finalized.\n\nStatistical Package for Social Sciences (SPSS version 21) was used for data entry and analysis. Descriptive analysis was used to record frequencies and percentages. A Chi-square test was used to determine the significant correlation between the dependent variables (pharmacists’ role and involvement in patient counseling, handling and reporting adverse effects of medications in patients, participation in health awareness programs, and pharmacists’ role in minimizing prescribing errors and identifying drug-related problems) and the independent variables (age and gender, type of pharmacy, and years of practice). A p-value of ≤ 0.05 was considered statistically significant at a 95% confidence interval for the differences.\n\n\nResults\n\nIn both qualitative and quantitative phases of the study, the age of participants ranged from 20 to 50 years, with a small number (n = 5) of older participants who were over 50 years old, as shown in Table 1, which presents the demographic profile of participants. No missing data were reported. In the qualitative phase, the thematic content analysis of the data obtained coded five main concerns, namely (a) patients reporting adverse drug events, (b) lack of patient counseling, (c) lack of participation in health awareness programs, (d) limitation in minimizing prescribing errors and identifying drug-related problems by pharmacists, (e) limitation in implementing pharmaceutical care services. The full dataset and transcripts can be found under Underlying data.\n\nAdverse drug reactions are among the negative consequences of therapy, as they affect the quality of life and well-being of patients. A large proportion of patients are admitted to hospital due to adverse drug reactions. A significant portion of this morbidity and mortality can be prevented through the use of pharmaceutical care. Almost all participants felt that patients did not inform pharmacists of adverse drug events. The following are participant responses relating to this concern:\n\nCommunity Pharmacist 1 (CP1) said “Because many patients do not know exactly what the pharmacist’s responsibilities are”\n\nPatients are not fully aware of pharmacists’ duties and trust physicians to discuss patient complaints because physicians are the first people called when patients feel unwell or have complaints.\n\nCP3 said “Patients do not inform the pharmacist because they do not even know that these effects are due to the drug itself”\n\nMost patients are unable to distinguish whether their symptoms are related to the medications they have been taking or to their disease or disease progression.\n\nHospital Pharmacist 2 (HP2) said “Sometimes patients come to the hospital and buy many medicines together without informing us about the side effects they are facing. We try to minimize this by informing them about the interactions between the drugs they buy and see if we can help them”\n\nPatients with polypharmacy are at high risk of side effects or drug interactions, most of which they are unable to recognise. They are unaware of this and require their physicians to change their prescriptions, usually without consulting the pharmacist to determine if there are specific interactions with other medications, foods, or even incorrect administration strategies.\n\nThe pharmacist’s responsibilities include efficiently counseling and educating the patient, suggesting the best medications, and showing the patient how to get the most benefit from therapy. As part of pharmaceutical care, the pharmacist must educate patients on the proper use, dosage, administration, side effects, drug interactions, and storage of medications. This includes respect for the patient, empathy, maintaining privacy during consultations, good communication skills, prompt dispensing, and providing all drug information. The Lack of pharmacists’ involvement in patient counseling is the greatest limitation to the pharmacist’s counseling role, which is considered the mainstay of his or her practice, and also contributes to his or her social underestimation.\n\nCP2 said “Most patients get advice from their doctor”\n\nPhysicians play an important role in patient counselling and education. Patients feel that they get what they need from their physicians, which has a significant impact on the pharmacist’s role in counselling.\n\nCP6 said “They do not fully understand the pharmacist’s role as a medicines expert and are more likely to trust the doctor”\n\nPatients want physicians to give them more medical advice about their conditions. They feel that this is what they wanted in the first place and that they are covered by their insurance and doctors should provide this for them.\n\nHP1 said “Issues of regulation and legislation, e.g. we are not allowed to dispense medicines without a doctor’s prescription or authorisation”\n\nThe influence of health authorities and regulations on the role of the pharmacist in the treatment of self-limiting diseases with over-the-counter medications (OTC) and the other diseases is based on prescription medications (POM) administered by physicians, where the role of the pharmacist is to dispense only the approved prescriptions.\n\nPharmacists play an important role in society. They are responsible for educating the public and spreading awareness about the rational use of medicines, as well as advising people about healthy diet and lifestyle. Such a role can be achieved through active participation in health education programs. Most pharmacists suffer from the fact that they rarely participate in such programs compared to other medical professions.\n\nCP4 said “Because of the regulations in the medical institutions that do not allow pharmacists to play their role”\n\nPharmacists believe medical facilities are more actively involved in health education programs and invite more physicians than pharmacists.\n\nCP8 said “Because of workload, lack of time and professional constraints”\n\nIn fact, there are several obstacles that limit the expansion of pharmacists’ activities outside their pharmacies, such as workload and staff shortages.\n\nHP6 said “Time management is important because most pharmacists are busy managing cases and inpatients”\n\nIn the hospital, pharmacists spend more time managing and using medications, which in turn affects their ability to pursue other activities. In fact, hospital pharmacists should have time to participate in continuing education programs.\n\nMedications today are complex not only in pharmacological terms, but also in terms of their administration and dispensing, as they undergo a long process ranging from prescription by the physician, dispensing and counseling by the pharmacist, and use by the patient or caregiver. Because of this complexity, there is a risk of medication errors or drug-related problems. Therefore, the role of pharmaceutical care is to minimize and prevent these errors. In most cases, pharmacists use their expertise in medications in conjunction with ongoing direct consultation with the physician to maximize patient benefit and safety by avoiding prescribing errors and identifying drug-related problems as much as possible.\n\nCP9 said “Pharmacists play a key role in reducing errors by reviewing prescriptions for drug interactions, dosing errors, contraindications, and other medication-related considerations”\n\nPharmacists are well positioned to play an important role in minimizing medication errors by reviewing medications prior to dispensing through a variety of processes, including double-checking strategy, patient reconciliation review, and prescription review based on the patient’s medical history.\n\nCP5 said “By properly counseling and informing patients about drug-food interactions”\n\nIdentification of drug-related problems and minimization of errors can be achieved through appropriate consultation between the patient and the pharmacist. Several points can be emphasized, such as the correct strategy and timing for taking medications, avoiding drug-food interactions, or drug-drug interactions.\n\nHP4 said “By calling the doctor and asking if they have provided the prescribed medication about the patient’s condition and if we can switch to a better medication”\n\nIn hospitals, communication between pharmacists and physicians can help achieve better patient outcomes by addressing untreated conditions and missing diagnoses and providing better options based on medications available in the hospital pharmacy.\n\nHP3 said “In this hospital, we report any error by putting it in writing and sharing it with the whole team at the end of each month so that each of us is aware of any possible error”\n\nHospital pharmacists effectively participate in reporting medication errors by going through several steps, namely: consistently reporting errors, collecting the reports monthly, analyzing the reports based on the nature of the errors, providing solutions on how to avoid them in the future, then sharing and discussing the errors and solutions in meetings with the hospital medical team to raise awareness and prevent such errors.\n\nThe implementation and adoption of pharmaceutical care can face many obstacles and barriers, such as lack of time due to workload, an insufficient number of qualified staff, lack of technology or skills in using technology programs, insufficient clinical and therapeutic knowledge, difficulty in accessing the patient’s medication and medical history, lack of communication skills, and lack of self-confidence. Other barriers include inadequate understanding of pharmaceutical care concepts and lack of patient and physician acceptance of pharmacist interventions. These barriers impede widespread adoption of this practice.\n\nPharmacists have been unable to develop a comprehensive practice due to numerous factors that continue to obscure their critical role and contribution to patient health.\n\nCP7 said “The role of the pharmacist is being replaced by other healthcare providers”\n\nPharmacists believe that several tasks that pharmacists can perform have been replaced or are already covered by nurses and physicians. We believe that other health care providers can participate in pharmaceutical care through interprofessional collaboration, but qualified pharmacists are best suited to assume these roles.\n\nCP2 said “Lack of opportunities for pharmacists”\n\nPharmaceutical care services are already widely used in health care systems. But it is not enough that these services exist; they should also be operated by qualified pharmacists. Therefore, appropriate positions for pharmacists should be created to successfully activate these services.\n\nHP5 said “Market’s needs, depends on what are the markets needs at each different place and time.”\n\nSome pharmacists believe that the implementation of pharmaceutical care is influenced by economics and markets. Such thoughts could be debunked if pharmaceutical care were properly implemented and presented as a strategy for cost-effective medication use. This leads to minimizing errors, using effective medications, and reducing drug costs, all of which have a major impact on hospital budgets.\n\nIn the quantitative phase, a total of 400 questionnaires were distributed. Of these, 305 pharmacists agreed to participate and completed the questionnaire (response rate 76.3%). The majority of the participants were between 20-40 years old (n = 258, 84.6%). The respondents were female (n = 159, 52.1%) and male (n = 146, 47.9%) pharmacists. More than half of the participants were Arab pharmacists (n = 177, 58%) and 42% (n = 128) were non-Arabs. Most participants worked in community pharmacies (n = 269, 88.2%) and to a lesser extent in hospital pharmacies (n = 36, 11.8%). Almost half of the participants (n = 136, 45%) had been practicing as pharmacists for less than five years but more than two years, while the others (n = 169, 55%) had been working as pharmacists for more than five years. Participants were from the seven largest cities in the UAE, Sharjah (n = 101, 32.9%), Ajman (n = 97, 31.8%), Abu Dhabi (n = 86, 28.2%), Dubai (n = 14, 4.6%), Ras Al-Khaimah (n = 3, 1%), Fujairah (n = 3, 1%) and Umm Al Quwain (n = 1, 0.3%). Table 1 shows the demographic profile of the respondents.\n\nPharmacists’ responses to the questions describing their interaction with physicians regarding patients’ medication showed different types of responses (Table 2). The majority of pharmacists (n =139, 46%, n = 135 43%) responded “sometimes” or “rarely-never” to the question of whether physicians consider their suggestions and input regarding medication therapy. When asked how often they were contacted by physicians regarding their patients’ prescriptions, they responded “rarely to never” (n = 179, 58.69%). The most frequently cited reasons for these contacts were drug alternatives (n = 194, 63.5%), drug availability (n = 168, 55.1%), and drug dosage (n = 152, 49.8%). Most pharmacists (n = 153, 50%) responded “sometimes” when physicians considered their suggestions about drug-related problems. Table 2 shows the interaction with physicians regarding patients’ medication.\n\nPatient counseling is as important as drug therapy and is considered a major component of pharmaceutical care practice. When pharmacists were asked if physicians were willing to accept their involvement in patient counseling, 43.6% (n = 133) responded positively while 56.4% (n = 172) responded negatively. There was a significant relationship between physicians’ willingness to accept pharmacists’ involvement in patient counseling and pharmacists’ years in practice (p < 0.001).\n\nThe majority of the pharmacists participated in patient education (n = 300, 98.6%), took adequate time for each patient (n = 246, 80.6%), informed patients about drug-drug or drug-food interactions (n = 240, 78.6%), instructed patients on the use of their medications (n = 282, 92.6%), informed patients about the side effects of medications (n = 200, 65.6%), and informed patients about proper storage conditions of medications (n = 243, 79.6%). The mean response regarding pharmacists’ approach to patient counseling was 77.1%. There was a significant association between the amount of time they spent with each patient and the type of pharmacy (p = 0.0083), education about side effects, and years of pharmacist experience (p = 0.0237). Table 3 illustrates the approach to patient counseling and pharmacists’ responses.\n\nThe current study also found that 59.7% (n = 182 \"mean\") of participants reported that their patients were aware of the pharmacist’s role in managing adverse drug reactions (ADRs). There was a statistically significant association between patients’ reports of adverse drug reactions and pharmacist age (p = 0.0216) and type of pharmacy (p = 0.0482). Table 4 shows the patients’ reports of adverse drug reactions that occurred and the pharmacists’ responses.\n\nParticipants were also asked about their active participation in health awareness programs and their responses were positive on average (n = 197.75, 64.8%). There was a statistically significant relationship between participation in health awareness programs and pharmacists’ age (p = 0.0441, p = 0.0002) and years in practice (p = 0.042). Table 5 shows the participation of participants in health awareness programs.\n\n* N/A: “not applicable”: it can’t be calculated.\n\nInterestingly, the positive average of participants was in terms of reducing prescribing errors and identifying drug-related problems (n = 28, 9.2%). There was a significant association between finding irrelevant drug prescriptions and pharmacist age (p = 0.0484). Table 6 shows participants’ perceptions of reducing prescribing errors and detecting drug-related problems.\n\nThe main barriers to implementing pharmaceutical care in the UAE cited by participants in this study were: Patients are not interested or in a hurry (n = 193, 63.3%), lack of time (n = 114, 37.4%), the professional role of pharmacists is not identified or clear (n = 99, 32.5%), communication barriers (n = 89, 29.2%), lack of financial benefits (n = 66, 21.6%), lack of scientific evidence for some medicines (n = 38, 12.5%), and inadequate expertise (n = 37, 12.2%). Figure 1 shows the barriers that prevent pharmacists from practicing pharmaceutical care for their patients.\n\n\nConclusions/Discussion\n\nPharmaceutical care is considered a new concept in the UAE. This concept has only recently been introduced in pharmacy schools. Therefore, the adaptation of pharmaceutical care practice is still in its infancy.\n\nBoth the qualitative and quantitative phases of the current study showed almost comparable results: Pharmacists are not convinced that their suggestions and contributions as a member of the health care team are taken into account by physicians.14 This type of discrepancy affects trust between the two professions and impacts pharmacists’ performance when they make suggestions because they feel their input is ignored. The study also found that there is a lack of communication between physicians and pharmacists regarding drug information, despite physicians’ recognition of pharmacists’ role as drug experts.15\n\nIn fact, the current results were consistent with several previous studies conducted in many Middle Eastern countries, including the UAE. A nationwide survey of final year pharmacy students in the UAE on attitudes and perceived barriers related to pharmaceutical care showed that the majority of pharmacists believe in the importance of pharmaceutical care services in improving patient health and advancing pharmacy practice. However, the role of the pharmacist is still poorly perceived and the lack of a consultation area or inappropriate pharmacy design were cited as the main barriers.16 In another study, the constant occurrence of medication dispensing errors was cited as a reason for introducing safe medication dispensing training programs.17\n\nAnother study pointed to the fundamental development of strategies to improve the quality of pharmacy services, aimed at supporting the maximum number of pharmacies, increasing the frequency of patient consultations and reducing the number of treatment errors.13\n\nThe consultative part of the interaction between the pharmacist and the patient is considered the core statement of pharmaceutical care practice. Thus, in his daily practice, the pharmacist must use his clinical knowledge, skills and attitude to provide the patient with a complete picture that includes information about the disease and every aspect of drug therapy, such as the use of the drug, its side effects, its interactions and storage conditions. A considerable number of pharmacists in this study are involved in patient counseling, but there are several barriers that prevent pharmacists from achieving a 100% counseling rate.\n\nThis may explain the lack of public confidence and education campaigns about the contribution of pharmacists to improving patient and public health. Given the limited statistical evaluation of such services in practice, the gradual introduction of an interprofessional education system in schools of medicine, pharmacy and nursing is a must. This will improve the existence of each medical profession in society while providing comprehensive and efficient patient-centred care. The impact of this new vision will contribute to an improved integration of pharmacists into the healthcare system.18,19\n\nAccording to the respondents in this study, not all patients are aware of the role of pharmacists in resolving adverse drug events, which is a barrier to the implementation of pharmaceutical care. Although pharmacists have been shown to share the most up-to-date evidence-based information from international sources such as the World Health Organization (WHO), the US Pharmacists Association, and the US Occupational Safety and Health Administration, and prevent crucial medication misuse or accidental loss.20–25\n\nIndeed, pharmacists have increased their knowledge of drugs and their side effects. At the same time, society chose pharmacists as the first direct source of up-to-date, trustworthy, and unbiased information. This frequent communication between pharmacists, patients and other health professionals strengthened the bond between individuals at the level of respect, trust, psychological support, information sharing and, above all, belief in the knowledge of each member as a dependent factor for the success of the whole scientific sector and the improvement of patient care.25,26\n\nHealth awareness programs are sometimes organized in hospitals but not in pharmacies, so a large proportion of pharmacists cannot participate in these programs. Almost 50% of pharmacists who participated in this study cannot participate in such programs because their workplace does not organize health awareness programs. 100% of the participants agreed that pharmacists should participate more in health awareness programs because it will help the society to know the role of pharmacists and thus strengthen their position in the health care system and develop the pharmacy profession in this country.\n\nAlthough this study found only a small percentage (9.2%) of positive perceptions in reducing prescribing errors and identifying drug-related problems, prescribing errors can be reduced if we use pharmacists’ drug knowledge and if pharmaceutical care practices are implemented correctly. Screening and correction of prescribing errors are considered one of the roles of pharmacists.27–31\n\nThe majority of the participants in this study (63.3%) believed that patients are either not interested in pharmaceutical care services or are in a hurry, so pharmacists let patients leave the pharmacy without introducing them to the idea of pharmaceutical care and without giving the patient enough information about their medications. These incorrect assumptions are due to the pharmacists’ lack of motivation and patients’ lack of awareness about the role of pharmacists.\n\nAll over the world, pharmacists are suffering from lack of time and increased workload. Therefore, pharmacists are unable to provide professional pharmaceutical care to every patient, which negatively affects patient satisfaction. This finding is consistent with a previous study conducted in UAE and other countries.32–37\n\nMost people in developing countries see pharmacists as drug dispensers because their interaction with pharmacists is limited to dispensing the prescription and medicines.38 Pharmacists should promote pharmaceutical care by practicing it with their patients until this practice becomes routine and expected when patients visit a pharmacy. This will help raise awareness of the role of pharmacists in the health care system and in society.8\n\nThese solutions should be supported by the health authorities as the adoption of this practice will have many positive effects on the health system of the country.\n\nAlthough participants were randomly selected from the different emirates of the UAE, the sample selection was not systematically distributed. In fact, we had difficulty recruiting participants because there were many barriers to active participation by pharmacists, such as lack of time and an insufficient number of supporting staff. In addition, the number of participants from hospital pharmacies was low because most pharmacists work in community pharmacies. The current study has several strengths: The study had a representative sample of pharmacists who were randomly selected, the technique used for data collection (face-to-face written questionnaires and appointment interviews) had a high response rate, and two methods of analysis were used. Therefore, the results of our study can be generalized to represent a broader population.\n\nThis study suggests that pharmacists working in the UAE do not implement pharmaceutical care as part of their daily routine work. However, pharmacists in the UAE have a positive attitude towards pharmaceutical care but lack consistency in delivering these services. Pharmacists are well placed to practice pharmaceutical care and they can provide it to patients when needed, but this is not enough. The health care system needs more pharmacist engagement. Pharmacists need to bridge the gap between them and physicians because they share a common goal to improve patient outcomes and quality of life. Health authorities should pay more attention to the role of pharmacists in the health system and help them to overcome the obstacles and barriers in the spread of pharmaceutical care to facilitate pharmaceutical care in practice and see the positive impact of this practice in all areas.\n\nThe major barriers faced by pharmacists in the UAE are patients’ ignorance of the pharmacist’s role, lack of time, and lack of motivation to implement this concept. Therefore, concerted efforts involving health authorities and educational institutions should be made to transform this profession from its traditional practice to a more patient-centered practice.\n\n\nData availability\n\nOpen Science Framework: Current views of community and hospital pharmacists on pharmaceutical care services in the United Arab Emirates: A mixed methodological study. https://osf.io/gzh8e\n\nThis project contains the following underlying data:\n\n‐ Full transcripts Qualitative interview.docx\n\n‐ Responses.xlsx\n\nThis project contains the following extended data:\n\n- Questionnaire.pdf\n\n\nReporting guidelines\n\n\n\n- COREQ_checklist F1000R.pdf\n\n- STROBE_checklist_cross-sectional score updated.doc\n\nData are available under the terms of Creative Commons Attribution 4.0 International License (CC-BY 4.0).",
"appendix": "References\n\nAwad A, Al-Ebrahim S, Abahussain E: Pharmaceutical care services in hospitals of Kuwait. World Health. 2006; 16: 18.\n\nAzhar S, Hassali MA, Taha A, et al.: Evaluation of the perception of community pharmacists regarding their role in Pakistan’s healthcare system: a qualitative approach. Trop. J. Pharm. Res. 2013; 12: 635–639.\n\nAzhar S, Hassali MA, Murtaza G, et al.: Current clinical practices in Pakistan and hospital pharmacist’s perception towards their role: a qualitative approach. Lat. Am. J. Pharm. 2012; 31.\n\nOlajide AS, Oladayo A, Emmanuel UI: Community pharmacy/hospital based assessment of clinical pharmacist intervention for safe and effective drug use. Journal of Applied Pharmaceutical Science. 2012; 2: 66–69. Publisher Full Text\n\nAbu Hagar R, El-Dahiyat F, El Refae G: Risk management in community pharmacy practice in Abu Dhabi Region: a cross-sectional study. J. Pharm. Health Serv. Res. 2020; 11: 275–285. Publisher Full Text\n\nAlslubi H, El-Dahiyat F: Patient safety practices among community pharmacists in Abu Dhabi, United Arab Emirates. J. Pharm. Health Serv. Res. 2019; 10: 203–210. Publisher Full Text\n\nRayes IK, Hassali MA, Abduelkarem AR: The role of pharmacists in developing countries: The current scenario in the United Arab Emirates. Saudi Pharm J. 2015; 23: 470–474. PubMed Abstract | Publisher Full Text\n\nReeves S, Macmillan K, van Soeren M : Leadership of interprofessional health and social care teams: a socio-historical analysis. J. Nurs. Manag. 2010; 18: 258–264. Publisher Full Text\n\nHealth: ADDo. Healthcare Providers Manual.Nov 2017. (accessed 25 October 2018).Reference Source\n\nJia P, Zhang L, Zhang M, et al.: Safety culture in a pharmacy setting using a pharmacy survey on patient safety culture: a cross-sectional study in China. BMJ Open. 2014; 4: e004904. PubMed Abstract | Publisher Full Text\n\nAzhar S, Hassali MA, Ibrahim MM: Perceptions of hospital pharmacist’s role in Pakistan’s healthcare system: a cross-sectional survey. Trop. J. Pharm. Res. 2011; 10. Publisher Full Text\n\nBond C, Raehl CL, Franke T: Clinical pharmacy services, hospital pharmacy staffing, and medication errors in United States hospitals. Pharmacotherapy: The Journal of Human Pharmacology and Drug Therapy. 2002; 22: 134–147. PubMed Abstract | Publisher Full Text\n\nMurtaza G, Kousar R, Azhar S, et al.: What do the hospital pharmacists think about the quality of Pharmaceutical Care Services in a Pakistani Province? A mixed methodology study. Biomed. Res. Int. 2015; 2015: 1–6. Publisher Full Text\n\nCrilly P, Patel N: Community Pharmacists’ Involvement in Research in the United Kingdom.2017; 5. Publisher Full Text\n\nIbrahim OM, Ibrahim R: Perception of physicians to the role of clinical pharmacists in United Arab Emirates (UAE). Pharmacology & Pharmacy. 2014; 05: 895–902. Publisher Full Text\n\nTawfiq AM, Alomar MJ, Hassan N, et al.: Nationwide survey on attitudes and perceived barriers toward provision of pharmaceutical care among final year undergraduate pharmacy students in the United Arab Emirates. PloS one. 2021; 16: e0246934. PubMed Abstract | Publisher Full Text\n\nIbrahim OM, Ibrahim RM, Meslamani AZA, et al.: Dispensing errors in community pharmacies in the United Arab Emirates: investigating incidence, types, severity, and causes. Pharmacy Practice (Granada). 2020; 18: 2111. Publisher Full Text\n\nBloomfield JG, Schneider CR, Lane S, et al.: Evaluating a large-scale introductory interprofessional education workshop for developing interprofessional socialisation in medical, nursing and pharmacy students: A quasi-experimental pre-test post-test study. Nurse Educ. Today. 2021; 99: 104777. PubMed Abstract | Publisher Full Text\n\nPharmacy ACoCPage RL, Hume AL, et al.: Interprofessional education: principles and application a framework for clinical pharmacy. Pharmacotherapy: The Journal of Human Pharmacology and Drug Therapy. 2009; 29: 879–879. Publisher Full Text\n\nSiddiqui MA, Abdeldayem A, Abdel Dayem K, et al.: Pharmacy leadership during emergency preparedness: Insights from the Middle East and South Asia. Am. J. Health Syst. Pharm. 2020; 77: 1191–1194. PubMed Abstract | Publisher Full Text\n\nPharmacists: ASoH-S. Assessment of evidence for COVID-19-related treatments: updated4/17/20.Accessed April 10, 2020.Reference Source\n\nMedicine. S.: Global COVID-19 prevention [video online].March 2020. Accessed April 10, 2020.Reference Source\n\nConvention: USP. USP response to shortages of garb and personal protective equipment (PPE) for sterile compounding during COVID-19 pandemic.March 2020. Accessed April 10, 2020.Reference Source\n\nConvention: USP. Compounding alcohol-based hand sanitizer during COVID-19 pandemic.March 2020. Accessed April 10, 2020.Reference Source\n\nPractices: IfSM. Special alert: medication safety alert!.March 26, 2020. Accessed April 10, 2020.Reference Source\n\nAdministration: OSaH. Guidance on preparing workplaces for COVID-19.March 2020. Accessed April 10, 2020.Reference Source\n\nChangulani T, Mustafa MZ, Ahuja S, et al.: Minimising prescription errors—a quality improvement project in the ophthalmology department in a tertiary referral hospital. Int. Ophthalmol. 2021; 41: 3041–3046. Publisher Full Text\n\nAnzan M, Alwhaibi M, Almetwazi M, et al.: Prescribing errors and associated factors in discharge prescriptions in the emergency department: A prospective cross-sectional study. PloS one. 2021; 16: e0245321. Publisher Full Text\n\nAlabid AHMA, Ibrahim MIM, Hassali MA, et al.: Community pharmacists’ awareness toward their roles in healthcare and interaction with general practitioners: A cross-sectional study. Journal of Pharmacy And Bioallied Sciences. 2021; 13: 220.\n\nVelo GP, Minuz P: Medication errors: prescribing faults and prescription errors. Br. J. Clin. Pharmacol. 2009; 67: 624–628. PubMed Abstract | Publisher Full Text\n\nPoudel R, Piryani R, Shrestha S, et al.: Prescription errors and pharmacist intervention at outpatient pharmacy of Chitwan Medical College. Journal of Chitwan Medical College. 2015; 5: 20–24. Publisher Full Text\n\nGhazal R, Hassan NAG, Ghaleb O, et al.: Barriers to the implementation of pharmaceutical care into the UAE community pharmacies. IOSR J Pharm. 2014; 4: 68–74.\n\nOkuyan B, Balta E, Ozcan V, et al.: Turkish community pharmacists’ behavioral determinants in provision of pharmaceutical care to elderly patients. Int. J. Clin. Pharm. 2021; 43: 1024–1035. Publisher Full Text\n\nFarha RA, Saadeh M, Mukattash TL, et al.: Pharmacy students’ knowledge and perception about the implementation of pharmaceutical care services in Jordan. Jordan Journal of Pharmaceutical Sciences. 2021; 14.\n\nUema SA, Vega EM, Armando PD, et al.: Barriers to pharmaceutical care in Argentina. Pharm. World Sci. 2008; 30: 211–215. PubMed Abstract | Publisher Full Text\n\nVan Mil J, De Boer W, Tromp T: European barriers to the implementation of pharmaceutical care. Int. J. Pharm. Pract. 2001; 9: 163–168.\n\nLoh P, Chua SS, Karuppannan M: The extent and barriers in providing pharmaceutical care services by community pharmacists in Malaysia: a cross-sectional study. BMC Health Serv. Res. 2021; 21: 1–14.\n\nAzhar S, Hassali MA, Ibrahim MIM, et al.: The role of pharmacists in developing countries: the current scenario in Pakistan. Hum. Resour. Health. 2009; 7: 1–6."
}
|
[
{
"id": "141859",
"date": "11 Jul 2022",
"name": "Hadeer Akram Abdulrazzaq Al-Ani",
"expertise": [
"Reviewer Expertise Clinical Pharmacy",
"Public Health",
"Biostatistics",
"Genetic pharmacology",
"and Pharmacoepidemiology."
],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nI would like to thank the authors for sharing their work in writing this manuscript. I have minor corrections which need to be edited/corrected. Ambiguous results should be detailed because everything should be clear to readers.\nPlease update all references within the last five years (2017-2022), especially those related to the problem statement, methodology, and discussion sections. The majority of references are outdated.\n\nIn Statistical Analysis: Please correct the Chi-square to find the association, not the correlation. “A Chi-square test was used to determine the significant correlation between..”\n\nResults. Rephrase “There was a statistically significant association between patients’ reports of adverse drug reactions and pharmacist age (p = 0.0216) and type of pharmacy (p = 0.0482)”. “There was a statistically significant relationship between participation in health awareness programs and pharmacists’ age (p = 0.0441, p = 0.0002) and years in practice (p = 0.042)”.\n\nResults. Table 3. Please give more explanation in relevant texts for the results which type of pharmacy (community or hospital) showed a higher amount of time. Same thing for informing the patients about drug side effects and years of experience. Need explanation about “Physicians are ready to accept the pharmacists’ participation” and years of experience.\n\nResults: Table 4. Please provide more details in relevant texts about which pharmacy showed a higher level of patient awareness of ADRs reporting.\n\nResults: Table 5. Please give more details in relevant texts about which age with hospital organizes health awareness programs and pharmacists' participation in organizations. Same for the experience with the hospital organizes health awareness programs and pharmacists' satisfaction.\n\nResults: Table 6. Please give more details in relevant texts about which age and years of experience are more levels of irrelevant drug prescriptions\n\nFigure 1. Please select another color than black (like white) for the percentage in bars. It is not clear.\n\nDiscussion: “The current results were consistent with several previous studies”. Which studies?\n\nSome sentences in the discussion are without references. Please add more references. Like “These incorrect assumptions are due to the pharmacists’ lack of motivation and patients’ lack of awareness about the role of pharmacists”\n\nIs the work 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": "8912",
"date": "01 Dec 2022",
"name": "Zelal Kharaba",
"role": "Author Response",
"response": "Thank you for taking the time to review our manuscript and for your valuable comments. Kindly find here our point-to-point reply to your comments: Please update all references within the last five years (2017-2022), especially those related to the problem statement, methodology, and discussion sections. The majority of references are outdated. Response: We would like to thank the reviewer for his time and efforts in critically reviewing our manuscript. Please note that References have been updated as per the advice, however, we kept few references- although outdated - as they are important for the idea of our article. In Statistical Analysis: Please correct the Chi-square to find the association, not the correlation. “A Chi-square test was used to determine the significant correlation between..” Response: Revised and highlighted in the text. Results. Rephrase “There was a statistically significant association between patients’ reports of adverse drug reactions and pharmacist age (p = 0.0216) and type of pharmacy (p = 0.0482)”. “There was a statistically significant relationship between participation in health awareness programs and pharmacists’ age (p = 0.0441, p = 0.0002) and years in practice (p = 0.042)”. Response: Revised and highlighted in the text. Results. Table 3. Please give more explanation in relevant texts for the results which type of pharmacy (community or hospital) showed a higher amount of time. Same thing for informing the patients about drug side effects and years of experience. Need explanation about “Physicians are ready to accept the pharmacists’ participation” and years of experience. Response: We would like to thank the author for his insightful comment which will improve our article. More explanation were added to the text as advised (highlighted in yellow). To detail the patient counselling approach (Table 3), there was a significant association between the amounts of time spent with each patient in community pharmacy compared to the hospital pharmacy. Additionally, pharmacists who practiced more than 10-years are significantly more engaged in patient counselling which reflect a higher physician acceptance rate as well as more patient education about side effects. Results: Table 4. Please provide more details in relevant texts about which pharmacy showed a higher level of patient awareness of ADRs reporting. Response: We would like to thank the reviewer for drawing our attention for this point. More explanation were added to the text as advised (highlighted in yellow). In detail, the community pharmacists showed significantly a higher level of patients’ awareness of adverse drug reaction reporting than hospital pharmacists. Results: Table 5. Please give more details in relevant texts about which age with hospital organizes health awareness programs and pharmacists' participation in organizations. Same for the experience with the hospital organizes health awareness programs and pharmacists' satisfaction. Response: We would like to thank the reviewer for drawing our attention for this point. More explanation were added to the text as advised (highlighted in yellow). In details, there was a significant difference among the older age pharmacists and the participation in health awareness program and the organization in such program. In addition, pharmacists who have more years of experience showed more satisfaction toward the health awareness programs. Results: Table 6. Please give more details in relevant texts about which age and years of experience are more levels of irrelevant drug prescriptions Response: We would like to thank the reviewer for drawing our attention for this point. More explanation were added to the text as advised (highlighted in yellow). In details, pharmacists with more years of practice showed to be more active in identifying any irrelative drug prescription. Figure 1. Please select another color than black (like white) for the percentage in bars. It is not clear. Response: Revised. Discussion: “The current results were consistent with several previous studies”. Which studies? Response: Revised and references added to this sentence. Some sentences in the discussion are without references. Please add more references. Like “These incorrect assumptions are due to the pharmacists’ lack of motivation and patients’ lack of awareness about the role of pharmacists” Response: Revised and references added."
}
]
},
{
"id": "141860",
"date": "15 Sep 2022",
"name": "Diana Malaeb",
"expertise": [
"Reviewer Expertise Clinical practice"
],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 mixed methodological study among hospital and community pharmacists across the UAE. The aim of the study is to explore the current views of pharmacists in the United Arab Emirates (UAE) on pharmaceutical care services and the nature of barriers encountered in practice using qualitative and quantitative assessment methods. The study has adequate power and used a validated tool in quantitative (n=305 pharmacist) and qualitative (n=15 interviewer pharmacists) approaches. However, there are few points that the authors need to clarify and elaborate more:\n\nThe title of the manuscript is explicit and covers the scope of the study. The method is appropriate, but the study need to include more number of hospital pharmacists to be consistent with the number of community pharmacists. I suggest this need to be clearly added in the limitation part of the study.\n\nThe seven criteria that the questionnaire was consisted of was wide enough to cover many important aspects in pharmaceutical care services as I think; these are 1) :vital statistics of the population, 2) pharmacist-physician interactivity, 3) pharmacist’s role and involvement in patient counseling, 4) handling and reporting of adverse drug reactions in patients, 5) participation in health awareness programs, 6) pharmacist’s role in minimizing prescribing errors and identifying drug-related problems, 7) barriers to implementing pharmaceutical care in the UAE. Authors need to briefly explain the selection of these seven barriers among other barriers and suggest few other barriers that future studies need to explore.\n\nThe response rate was 76% (305/400 invited pharmacists)! Authors need to give reasons for the low response rate.\n\nThere is one table that describes the demographics. However, I did find it very interesting that the number of participants aged>50 is only 5 participants, can the authors elaborate more on that?\n\nI see that there are statistical differences in table-4 (Patients inform the pharmacists about ADRs occurrence and ask about how it can be managed---but does that translate into actual differences? Does it?\n\nThe finding in table-4 (Patients are aware of ADRs reporting) need to be discussed more in the discussion part. How can you explain the type of pharmacy and patients awareness of ADR?\n\nI enjoyed reading the manuscript and found it interesting--- but was surprised about the barriers to implementing pharmaceutical care in the UAE! 63.3% of patients think they are not interested or in a hurry and 32% of them thought that the professional role of pharmacists is not identified or clear. I think the authors covered these points in the discussion but they need to explain more why these two barriers existed and suggest solutions for them.\n\nIs the work 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": "8913",
"date": "01 Dec 2022",
"name": "Zelal Kharaba",
"role": "Author Response",
"response": "Author: This is a mixed methodological study among hospital and community pharmacists across the UAE. The aim of the study is to explore the current views of pharmacists in the United Arab Emirates (UAE) on pharmaceutical care services and the nature of barriers encountered in practice using qualitative and quantitative assessment methods. The study has adequate power and used a validated tool in quantitative (n=305 pharmacist) and qualitative (n=15 interviewer pharmacists) approaches. However, there are few points that the authors need to clarify and elaborate more: The title of the manuscript is explicit and covers the scope of the study. The method is appropriate, but the study need to include more number of hospital pharmacists to be consistent with the number of community pharmacists. I suggest this need to be clearly added in the limitation part of the study. Response: We would like to thank the reviewer for her time and efforts in critically reviewing our manuscript. Please note that the suggested comment is added to the limitation part of the study and highlighted in yellow. The seven criteria that the questionnaire was wide enough to cover many important aspects in pharmaceutical care services as I think; these are 1) :vital statistics of the population, 2) pharmacist-physician interactivity, 3) pharmacist’s role and involvement in patient counseling, 4) handling and reporting of adverse drug reactions in patients, 5) participation in health awareness programs, 6) pharmacist’s role in minimizing prescribing errors and identifying drug-related problems, 7) barriers to implementing pharmaceutical care in the UAE. Authors need to briefly explain the selection of these seven barriers among other barriers and suggest few other barriers that future studies need to explore. Response: We would like to thank the reviewer for her time and efforts in critically reviewing our manuscript. Please note that the selection of the previous aspects was after an extensive literature review of many articles that deeply discussed many aspects in pharmaceutical care services. These aspects were gathered in the study questionnaire which was validated and piloted. We think that future studies need to explore these barriers in line with COVID-19 pandemic. In addition, future studies need to focus also on te effective participation of hospital pharmacists in reporting medication errors by going through several steps, namely: consistently reporting errors, collecting the reports monthly, analyzing the reports based on the nature of the errors, providing solutions on how to avoid them in the future, then sharing and discussing the errors and solutions in meetings with the hospital medical team to raise awareness and prevent such errors. The response rate was 76% (305/400 invited pharmacists)! Authors need to give reasons for the low response rate. Response: We would like to thank the reviewer for her time and efforts in critically reviewing our manuscript. In fact the low response rate among pharmacists is attributed to their busy schedule, the unavailability of sufficient number of pharmacists and the lack of appropriate time. There is one table that describes the demographics. However, I did find it very interesting that the number of participants aged>50 is only 5 participants, can the authors elaborate more on that? Response: In fact, UAE considered as a youth country with more than 77% of its population aged less than 55 years of age according to the statistics center 2022 (https://www.globalmediainsight.com/blog/uae-population-statistics/). This is applied for the pharmacy sectors as well. I see that there are statistical differences in table-4 (Patients inform the pharmacists about ADRs occurrence and ask about how it can be managed---but does that translate into actual differences? Does it? Response: We would like to thank the reviewer for her time and efforts in critically reviewing our manuscript. In fact this is translated into actual references as 62% of participants received patients report of ADRs. The finding in table-4 (Patients are aware of ADRs reporting) need to be discussed more in the discussion part. How can you explain the type of pharmacy and patients awareness of ADR? Response: We would like to thank the reviewer for her time and efforts in critically reviewing our manuscript. Table 4 shows the patients' reports of adverse drug reactions that occurred and the pharmacists' responses. In detail, the community pharmacists showed significantly a higher level of patients’ awareness of adverse drug reaction reporting than hospital pharmacists. I enjoyed reading the manuscript and found it interesting--- but was surprised about the barriers to implementing pharmaceutical care in the UAE! 63.3% of patients think they are not interested or in a hurry and 32% of them thought that the professional role of pharmacists is not identified or clear. I think the authors covered these points in the discussion but they need to explain more why these two barriers existed and suggest solutions for them. Response: We would like to thank the reviewer for her time and efforts in critically reviewing our manuscript. We think that the main cause of that is related to the long time consumed by patients in the medical centers and hospitals which affects the pharmacists’-patients’ discussion time. We suggest that the waiting time in health care centers need to be reduced and more time for the patients to spend with the pharmacists."
}
]
}
] | 1
|
https://f1000research.com/articles/11-694
|
https://f1000research.com/articles/11-56/v1
|
18 Jan 22
|
{
"type": "Research Article",
"title": "Measuring the motives of informal entrepreneurs",
"authors": [
"Noor Shahaliza Othman",
"Govindan Marthandan",
"Kamarulzaman Ab Aziz",
"Govindan Marthandan",
"Kamarulzaman Ab Aziz"
],
"abstract": "Background - Handling non-observed activities pose major challenges to the governments and other stakeholders. Non-observed activities refer to underground activities, illegal activities, informal sector and any other activities that result in goods or services consumed by the household. The impact of these non-observed activities shows that the volume of people involved in the informal sector will rapidly increase. Informal economic activities are technically illegal yet are not intended as antisocial,\n\nthereby remaining acceptable to many individuals within the society. This research aimed to identify the factors that lead to entrepreneurial necessity and opportunity.\n\nMethods – The data of 51 respondents who were employed as informal entrepreneurs in Klang Valley areas in Malaysia was collected with the use of a questionnaire and convenient and proportionate sampling techniques. The data were analysed using SPSS software.\n\nResults – The two primary drivers of informal entrepreneurial activity were necessity and opportunity. The inability to find a formal job was an example of being driven by necessity. Meanwhile, individuals that are driven by opportunity chose to work independently in these informal sectors. Between necessity and engagement, refinement acted as a mediator. Often, necessity and opportunity do not automatically translate into successful entrepreneurship; further refinement is required in terms of market potential, technology usage, location preferences, and capital requirements. Improved refinement results in increased entrepreneurial engagement. Conclusions - The role and contribution of the informal sector entrepreneurship in economic development need to be evaluated and not just observed as an opportunity for individuals who choose this type of career. Therefore, further research is required in a wider variety of contexts to evaluate whether the same remains true in different populations. The results of this study can be useful for the government to set policies to encourage the transition of informal to formal entrepreneurships in Malaysia.",
"keywords": [
"Necessity driven",
"Opportunity driven",
"Motives",
"Informal entrepreneur"
],
"content": "Introduction\n\nNon-observed activities typically include underground, illegal, and any other informal sector activities that results in commodities or services that a household can consume1. As a result of these unregulated activities, the informal sector of the population has increased significantly in recent years. In addition, while informal economic activities are technically illegal, they are not “antisocial in intent,” making them acceptable to a large segment of society despite being illegal.1\n\nThere has been many competing theories proposed to explain the rise of informal sector entrepreneurship. However, only a handful of people have attempted to document these divergent points of view, and even fewer have attempted an objective assessment of their validity. According to previous research, two major factors significantly impacted entrepreneurs in the informal sector. Push factors, also known as factors of necessity, and pull factors, also known as factors of opportunity.2–4\n\nAccording to Williams, C. C.,3 push factors can be understood as unemployment, underemployment, and dissatisfaction with current employment. “Necessity” entrepreneurs were forced into entrepreneurship due to a lack of alternatives. These are defined as a desire for independence, self-actualisation, financial benefits, and a desire to achieve a better balance between family and work obligations. Entrepreneurs seize such an “opportunity” because they want to be self-sufficient or own a business.2\n\nIndividuals who consider entrepreneurship as the best available option rather than the best option for them personaliy are referred to as necessity refinement types of individuals.5 Opportunity refinement implies to opportunity creation as opportunities usually do not matterialise unexpectedly and therefore must be created. Based on personal “rationale” and “talent”, an individual must decide whether or not to take advantage of the opportunity and select a preferred career path and industry. In actuality, ensuring that entrepreneurs have access to opportunities contributes to economic growth.\n\nThe number of informal entrepreneurs in Malaysia is on the rise yearly.6 The hardship and challenges of surviving in the current economic situation compels people to be inclined to engage in types of work that are usually of the informal business type other than their official work.\n\nLike formal and corporate entrepreneurs, informal entrepreneurs foster an informal mode of entrepreneurship in more diverse and creative sectors, which eventually it can significantly contributes to the national economy.\n\nAs such, the main objective of this paper is to empirically test the factors that create the need for entrepreneurial necessity and opportunity. The research findings are expected to shed light on why people choose informal entrepreneurship and how they become involved in these types of work over time. As a result, the government will be able to use this information when developing policies on entrepreneurship.\n\nThe informal sector provides substantial subsidies to certain economies, most notably those in developing countries.7 Consequently, this is critical for creating job oppertunities and income generation. For example, in 2005, analyses of the mixed-income of Malaysian households revealed that the informal sector contributed 13% to the country's gross domestic product.8 Although the previous study established that informal sectors are highly competitive andindividualized, the topic of informal sector disputes and competitiveness has had a relatively limited attention in the literature.9,10\n\nThe Organisation for Economic Co-operation and Development (2002)11 and the System of National Accounts (1993)12 have defined the informal sector as “an enterprise that includes households that produce goods solely for personal consumption, thus encompassing all units engaged in productive activities.”\n\nThe 15th International Conference of Labour Statisticians (1993)13 has also defined the informal sector as:\n\n(a) Characterised. The informal sector can be depicted rather well. These units are frequently associated at a low level, with little or no division in the middle of work and capital as production components, and on a small scale.\n\n(b) The distinctive characteristics of household businesses. The altered and unique possessions utilised do not have a place in the production units, at least not in accordance to their managers. The owners are required to increase the necessary finance at their own risk.\n\nAccording to the Malaysian Department of Statistics (2012),6 an employed person in the informal sector is defined as a working population in an establishment that meets the following conditions:\n\n(a) The business is not registered with the Malaysian Companies Commission or other professional organisations, including the local government\n\n(b) All or no less than one product or administration created are implied to be purchased or deal transaction.\n\n(c) The number of people employed is fewer than 10, and the company is not incorporated under a specific type of national legislation.\n\n(d) The establishment is onvolved in non-agricultural activities.\n\nWhile most informal entrepreneurs are found to capitalise on business possibilities, others are formed due to the owner's inability to obtain an adequate job or be obliged to work. When the two factors are examined, it is observed that in Africa, opportunity driven firms are more efficient and widespread than in other parts of the world.14\n\nOriginally, Schumpeter's definition of an entrepreneur was someone who is willing to take risks to capitalise on an existing business opportunity and who will start a new business if the idea is good and the opportunity exists.15 However, in developing countries, most businesses are started not because of opportunities but because the owners are unable to find employment in their desired field.\n\nAccording to the 2009 World Bank Enterprise survey on informal business in Cote d'Ivoire, Madagascar, and Mauritius, 39% of businesses were started by business owners who own the majority of the business but were unable to find job satisfaction.16 The remaining 61% took advantage of the opportunity to start their businesses or expand existing ones. Based on thisstudy, opportunities versus needs regarding business or informal entrepreneurship were related to a business's structure, performance, and problems encountered during operation.\n\nWhen it comes to running a business, opportunistic entrepreneurs may be more motivated and skilled than other types of entrepreneurs. Nonetheless, entrepreneur must always be prepared to encounter difficulties while conducting business. Typically, informal businesses operate on a small scale and engage in simple business activities.\n\nThe widely held belief is that informal sector entrepreneurs in developing countries are primarily necessity-driven individuals forced into entrepreneurship as a means of survival due to the absence of other options.5 Entrepreneurs in the informal sector can benefit from understanding the distinction between opportunity and necessity entrepreneurs. This distinction can be used to analyse acceptable entrepreneurs and better understand the thought processes of entrepreneurs in the informal sector.\n\nUntil recently, individuals who work entirely or partially in the informal sector were assumed to be motivated by necessity, pushed into this line of work in the absence of other options.\n\nAccording to William (2007),17 “Choose to participate in the informal economy because they find more autonomy, flexibility, and freedom in this sector than in the formal one. In other words, participants have the freedom of operating their own business; they have elasticity in defining hours or days of operation; they can use and develop their creativeness.”\n\nNonetheless, over the last decade or so, the opposite has begun to be discussed. Scholars have begun to refer to them as opportunity entrepreneurs, emphasising the word entrepreneur in the preceding statement. Despite the widespread belief that external pressures (such as economic reform, unemployment, and discrimination) drive people into the informal economy, the majority of the fifty informal sector entrepreneurs has discussed did so voluntarily.18,19 The majority of chance entrepreneurs came to this sector searching for a new career. Even individuals who began as necessity-driven entrepreneurs are more likely to develop a long-term commitment to their informal sector businesses due to the official economy's limited opportunities.\n\n\nMethods\n\nThis research adapted positivism discipline as a critical element. This quantitative study involved the collection of data via a structured questionnaire (Underlying data).20\n\nQuestionnaires were specifically prepared by using 5-point Likert-scale: (1) Strongly disagree – (5) Strongly agree, to reflect the agreement of respondents. The questions were also translated into the Malay Language to cater to the responders language preferences. The type of data used was primary data. The unit of analysis was an individual who was an informal entrepreneur. In this cross-sectional study, data was collected personally via face-to-face interview. Joe F. Hair et al.21 recommended 30 to 100 for the minimum sample size to provides valid results using SPSS Software (Version 26). Based on the G Power test, the sample size needed was 45 based on effect size and the number of predictors in the research. As a result, it was agreed that 100 questionnaires would be distributed via convenient sampling techniques to individuals who were employed as informal entrepeneurs in Klang Valley areas in Malaysia. This particular area was selected because it had the highest number of employed in the informal sector as reported by the Department of Statistics.6 However, only 51 of the 100 questionnaires distributed was used for further analysis. This was because at the screening and cleaning stage, 35 respondents were rejected due toincomplete or missing responses, additionally some respondents had registered businesses, and 16 had operated their businesses for less than a year.\n\nThe first section of the questionnaire asked respondents about their history of gross household income, the employment status and employment histories of household members, their ages, gender, and the type of work they relied on the most to maintain their standard of living (Underlying data).20 The following section contained an open-ended questions about whether they were self-employed or built their business - if they had built their business, they had to answer when these enterprises began, whether they conducted some or all of their transactions in the informal sector and various reasons for starting this venture.\n\nBelow are the research framework adopted for this study (Figure 1).\n\nAdapted from Ref. 20.\n\nPrior to conducting this study ethical approval was obtained from the Research Ethical Committee (Approval Number: EA2542021) of Multimedia University. Participants provided informed oral consent to participate in the study. Oral consent instead of written consent was obtained because of time constraints and illiteracy of some participants.\n\n\nResults\n\nAccording to this survey, 53% of the respondents are male and 47% are female. The characteristics of respondents is shown in Table 1.\n\nAccording to the results, 65% of respondents were married between the ages of 25 and 39. The committed married couples had mentioned that in order to provide for their families, they will take on any task. On the other hand, 41% of them earned less than RM1000 per month and had only completed secondary school Based on these findings education has a strong correlation with income. Therefore, it is critical to acquire a high degree of education to earn a higher salary. According to the demographic profile of the respondents, their competence and productiveness were in the medium range, which is consistent with previous studies. The vast majority of them conducted their business in person with the customers.They interacted directly with customers, and some operateed their businesses from their homes.\n\nAccording to Table 2, 53% of respondents identified themselves as “employed” informal entrepreneurs due to the opportunity-driven environment. From this list of opportunity-driven alternatives, most respondents (48%) choose to be informal entrepreneurs due to the time flexibility and preference for self-employment. That was the primary reason for setting up a business. Additionally, making time for other commitments such as family commitments, was important., Thus, time flexibility was one of the most critical criteria for informal entrepreneurs.\n\nAnother factor was family tradition, which accounted for 19% and past business experience, which accounted for 11% of the votes. However, necessity-driven factors such as the need to earn additional income to support their family, dissatisfaction with previous employment, the need to supplement net income, and an inability to find suitable jobs were cited as reasons for starting their businesses by 47% of informal entrepreneurs.\n\n\nDiscussion\n\nIn this study it was important to understand the reasons behind individuals pursuing informal business ventures, and whether these reasons are motivated by necessity or a sense of opportunity. Additionally, education is critical in guiding informal entrepreneurs. As a result, informal sector entrepreneurship should be reinterpreted as typically driven by opportunity, however its role in economic development should be examined. Additional studies are needed to challenge the image of entrepreneurship in the informal sector by assessing factors such as the level of education or political stance in certain communities, the rigidness of transitioning from informal to formal business, and also the policies available to harness this large area of entrepreneurship on a global scale.\n\n\nConclusions\n\nAs this is a cross-sectional study, the data cannot be utilised to establish a solid causal relationship based on the study's findings. As a result, a longitudinal studies are needed to examine the correlations between the study variables. In this study, which was prone to methodological bias, self-report data were utilised to create connections between variables. To minimise bias in future studies, closed envelopes should be used to guarantee confidentiality and anonymity throughout the data gathering process. Some respondents may underreport their informal entrepreneurial motivations out of concern for the implications of future legal actions on their business. This could result in reporting bias, which would have an effect on the study's results. To mitigate this, each study participant should be informed clearly about the study's goal and ensured that the information would be kept anonymous.\n\n\nData availability\n\nFigshare: Measuring the motives of informal entrepreneurs https://figshare.com/s/c70b053eacb5ac8a24e4.20\n\nThis project contains the following underlying data:\n\nData file 1. Questionnaire\n\nhttps://figshare.com/s/b9433876684c7fd89572.22\n\nThis project contains the following underlying data: Data file 1. MiniFund Questionnaire Report\n\nData are available under the terms of the Creative Commons Zero “No rights reserved” data waiver Attribution 4.0 International (CC BY 4.0).\n\n\nCompeting interests\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThis study was funded by the Ministry of Higher Education Malaysia. Grant number: FRGS MMUE/140011.\n\n\nAuthor contributions\n\nNoor Shahaliza Othman, Govindan Marthandan and Kamarulzaman Ab Aziz were involved in overall direction and planning and supervised the work. Noor Shahaliza developed the research framework, carried out the implementation and analysed the data. Govindan Marthandan and Kamarulzaman Ab Aziz advised on the execution process including supervised the progress.",
"appendix": "References\n\nWebb JW, Bruton GD, Tihanyi L, et al.: Research on entrepreneurship in the informal economy: Framing a research agenda. J. Bus. Ventur. 2013; 28: 598–614. Publisher Full Text\n\nAfrica S, Africa S: Retheorising the Motives of Informal Ent.2007.\n\nWilliams CC: Beyond necessity-driven versus.2008; 9(3): 1–10.\n\nBora RS: Migrant Informal Workers: A Study of Delhi and Satellite Towns. Mod. Econ. 2014; 05(05): 562–579. Publisher Full Text\n\nWilliams CC, Youssef Y: Is Informal Sector Entrepreneurship Necessity- or Opportunity-driven ?. Some Lessons from Urban Brazil. . 2014; 3(1): 41–53. Publisher Full Text\n\nDepartment of Statistics: Informal sector workforce survey report. Kuala Lumpur: Department of Statistics, Malaysia; 2012.\n\nGerxhani K: The informal sector in developed and less developed countries: a literature survey. Public Choice. 2004; 120(3): 267–300. Publisher Full Text\n\nBaharudin N, Othman M, Waty P, et al.: Informal Employment in Informal Sector Enterprises In Malaysia Abstrak. Jabatan Statistik Malaysia.2006.\n\nBeyer A: Motivations for Engaging in Entrepreneurial Activity in the Informal Sector in Sub Saharan Africa.2018.\n\nBrouwer S, Krol B, Reneman MF, et al.: Behavioral Determinants as Predictors of Return to Work after Longterm Sickness Absence: An Application of the Theory of Planned Behavior. J. Occup. Rehabil. 2009; 19(2): 166–174. PubMed Abstract | Publisher Full Text\n\nOrganisation for Economic Co-operation and Development Staff: Education at a glance: OECD indicators 2002. Paris: OECD; 2002.\n\nInter-Secretariat Working Group on National Accounts: Brussels/Luxembourg, New York, Paris, Washington, D.C., Pok, C. a frontera de la 1993.1993.\n\nHussmanns R: Statistical definition of informal employment: Guidelines endorsed by the Seventeenth International Conference of Labour Statisticians (2003). 7th Meeting of the Expert Group on Informal Sector Statistics (Delhi Group). 2004, February; (pp. 2–4).\n\nAmin M: Necessity vs. Opportunity Entrepreneurs in the Informal Sector.2008.\n\nSchumpeter JA: Entrepreneurship as innovation. University of Illinois at Urbana-Champaign's Academy for Entrepreneurial Leadership Historical Research Reference in Entrepreneurship. 2000.\n\nWilliams CC, Kedir AM: Explaining cross-country variations in the prevalence of informal sector competitors: lessons from the World Bank Enterprise Survey. Int. Entrep. Manag. J. 2019; 15(3): 677–696. Publisher Full Text\n\nWilliams CC: Entrepreneurs operating in the informal economy: necessity or opportunity driven?. J. Small Bus. Entrep. 2007; 20(3): 309–319. Publisher Full Text\n\nSnyder JM, Yackovlev I: Political and Economic Determinants of Changes in Government Spending on Social Protection Programs.2000.\n\nAl-Mataani R, Wainwright T, Demirel P: Hidden Entrepreneurs: Informal Practices within the Formal Economy. Eur. Manag. Rev. 2017; 14(4): 361–376. Publisher Full Text\n\nOthman NS, Marthandan G, Aziz KA: Measuring the motives of informal entrepreneurs.2021. Reference Source\n\nHair JF, Matthews LM, Matthews RL, et al.: PLS-SEM or CB-SEM: Updated Guidelines on Which Method to Use. International Journal Multivariate Data Analysis. 2017; 1(2): 107–123. Publisher Full Text\n\nOthman NS, Marthandan G, Aziz KA: Measuring the motives of informal entrepreneurs.2021. Reference Source"
}
|
[
{
"id": "120473",
"date": "17 Feb 2022",
"name": "Gurel Cetin",
"expertise": [
"Reviewer Expertise entrpreneuship"
],
"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 looks into an important area of informal entrepreneurship, but does not explain how are they different t formal entrepreneurs. Moreover, \"between necessity and engagement, refinement acted as a mediator\" - there is no finding or analysis concerning refinement as a mediator. Please explain.\n\nThe sample is too small, particularly for a SEM study. The authors tried to justify this but it is not really acceptable even if a normal distribution is assumed. King valley should be justified. Why it has been selected? How representative is it? Why not other sites?\n\nThe literature is somewhat weak concerning the theoretical background. Push and pull factors of entrepreneurship are also described in \"Alrawadieh, Z., et al. (2021). The interface between hospitality and tourism entrepreneurship, integration and well-being: A study of refugee entrepreneurs.\" Please refer to this paper.\n\nThe scales and measures used in the study should be described and justified.\n\n\"The following section contained open-ended questions about whether they were self-employed or built their business\" - These are not really exclusive since someone can be both self-employed and build their business.\n\nThe discussion and conclusions should be elaborated to include theoretical and practical contributions.\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? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nPartly\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? No",
"responses": []
},
{
"id": "125700",
"date": "04 Mar 2022",
"name": "Muhammad Ashraf Fauzi",
"expertise": [
"Reviewer Expertise Knowledge Management",
"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\nThank you for providing the opportunity to review this work. These are my comments:\nThe last sentence in the first para \"In addition, while informal economic activities are technically illegal, they are not “antisocial in intent,” making them acceptable to a large segment of society despite being illegal.\" - This sentence requires more explanation as it seems hanging. What does society correspond to it being illegal?\n\nSome of the paragraphs are too short. Example \"Like formal and corporate entrepreneurs, informal entrepreneurs foster an informal mode of entrepreneurship in more diverse and creative sectors, which eventually it can significantly contributes to the national economy\" - This paragraph should be combined with the previous.\n\nThere are some spelling mistakes \"onvolved\" for example in page 4. Another is \"thisstudy\".\n\nThe discussion is not enough. Please provide evidence and support from past studies that are in congruence with the author's findings.\n\nThe conclusion is actually a limitation, please reiterate and conclude why informal entrepreneurship is important to the society/nation-building, etc.\n\nThe manuscript requires proofreading, as there are grammar mistakes, spelling and other minor errors.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? 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/11-56
|
https://f1000research.com/articles/11-330/v1
|
18 Mar 22
|
{
"type": "Review",
"title": "p53 Mutation as Plausible Predictor for Endocrine Resistance Therapy in Luminal Breast Cancer",
"authors": [
"Freda Halim",
"Yohana Azhar",
"Suwarman Suwarman",
"Bethy Hernowo",
"Yohana Azhar",
"Suwarman Suwarman",
"Bethy Hernowo"
],
"abstract": "Endocrine therapy resistance in Luminal Breast Cancer is a significant issue to be tackled, but currently no specific biomarker could be used to anticipate this event. p53 mutation is widely known as one of Breast Cancer’s most prominent genetic alterations. Its mutation could generate various effects in Estrogen Receptor and Progesteron Receptor molecular works, tangled in events leading to the aggravation of endocrine therapy resistance. Hence the possibility of p53 mutation utilization as an endocrine therapy resistance predictive biomarker is plausible. The purpose of this review is to explore the latest knowledge of p53 role in Estrogen Receptor and Progesteron Receptor molecular actions thus aggravating the Endocrine Therapy resistance in Luminal Breast Cancer, from which we could define possibilities and limitations to utilize p53 as the predictive biomarker of endocrine therapy resistance in Luminal Breast Cancer.",
"keywords": [
"p53",
"predictor",
"endocrine therapy resistance",
"luminal breast cancer"
],
"content": "Introduction\n\nEndocrine Therapy (ET) resistance in Luminal Breast Cancer (BC) is a concerning issue. Approximately 30-40% of Luminal BC are ET resistant, which leads to a higher recurrence rate and worsened prognosis. Although it has been extensively studied, till now there is no single predictive biomarker has been established to predict which patient will develop ET resistance during the 5-years-course of endocrine therapy.1–3\n\nSuch predictive biomarker will be advantageous both for clinician and patients, as patients which probably have bigger chance for endocrine therapy resistance could be monitored closely. Perhaps later in the future it could help to effectively change the course of the therapy before recurrence is established (and it becomes too late), as well as to help clinicians to identify which patients will not have ET benefit in the first place.1,2 And as we know, current trend in clinical trials of BC treatment are moving into personalized and tailored therapy for each cases, therefore finding predictive biomarker to predict the ET resistance will be important issue for such theurapeutic program development as well.4\n\nEndocrine therapy resistance is complex molecular process which involved many process to develop. Several hypotheses has been developed regarding addressing such process, and finding such predictive biomarker. The resistance could develop at the start of the endocrine therapy (de novo or intrinsic resistance) or develop later in the course of the endocrine therapy. The hypotheses are ranging from the loss of hormonal receptor (HR) caused by ESR1 gene mutation and epigenetic mechanism,5–8 altered expression of co-factors (such as NF-kB, AIB1, SRC-1),9–11 crosstalk between ER and growth factors signaling (such as Her2neu, Insulin-like growth factor-1 receptor (IGF-1R))4,6,8,10,12,13 absent or reduced expression of negative regulator such as p21 and p27,14–16 metabolic resistance caused by polymorphism or loss of CYP2D6 (main enzymes responsible for converting tamoxifen into its active metabolites),2,4,8,10,17,18 NF1 mutation lead to MAPK pathway activation,19–24 APOBEC mutation associated with PI3KCA mutation,25 as well as tumor microenvironment roles such as NF-kB and TNF-α.9,11,26–29\n\nThe molecular mechanism of Estrogen Receptor (ER) and Progesterone Receptor (PR) actions are studied extensively for their association with ET resistance in Luminal BC. These molecular mechanisms additionally become an essential basis in rationalizing treatments such as Cyclin-CDK (Cyclin-Dependent Kinase) inhibitor and PI3K/Akt/mTOR inhibitor, which have been internationally accepted as current adjuvant treatments for Luminal BC with recurrence after ET resistance. Their actions therefore are basic and fundamental knowledge to find a logical explanation of endocrine therapy resistance, and most of the hypotheses above could be explained by the disruption of the ER and PR mechanism of actions, resulting increased cellular proliferation and decreased apoptosis.4,6,8–16,27,30\n\np53 mutation is one of the most frequent genetic alterations in BC, found in approximately 28.3%-35% of overall BC patients, with higher incidence in Luminal B BC (30-55%), Her-2neu overexpressive (70%) and TNBC group (80%).31–33 p53 mutation in hormonal positive BC will result in distinct poor prognosis, and especially seen in Luminal B BC with higher frequency and stronger association to poor prognosis compared to Luminal A BC.32,34 The mutation of this profound tumor suppressor gene may occur at the early onset of Luminal BC or progressively at the later course of the disease due to cancer cells’ ability to form more mutations in the advanced stage.20,31,35–37\n\np53 mutation has been known for more than four decades and its extensive roles span from cell cycle regulation, DNA repair, apoptosis process, cell metabolism, as well as immune response in tumor microenvironment.21,37–41 This versatile tumor suppressor gene has been studied in many cancers including breast cancer, and its protein accumulation as well as its mutation is conclusively found in numerous endocrine resistance breast cancer studies.20,35,36,42,43\n\nThis review will explore the current knowledge of ER and PR molecular mechanisms and their impact in initiating ET resistance in Luminal BC. Furthermore, we will discuss the apparent effect of p53 mutation in their molecular mechanisms, consequently aggravating ET resistance.\n\n\nEstrogen and estrogen receptor\n\nEstrogen is a steroid hormone that acts in several tissues such as skin, liver, bone, and breast. In breast tissue, Estrogen’s potent mitogenic effect will generate breast epithelial proliferation, alveolar growth, fat deposition, and fibrous tissue development during puberty, pregnancy, and lactation phases. These distinctive changes in the breast are affected by Estrogen, which works alongside Progesterone and other growth factors.44\n\nThe active form of Estrogen in breast tissue, Estradiol, and its metabolites have been acknowledged as essential factors of early malignant transformation, such as DNA single strand breaks and chromosomal impairment. Furthermore, it may lead to uncontrolled cell proliferation, accompanied by the development of cellular signalling collaborating in the cancerous cells’ progression. All events mentioned above will benefit the growth of cancer cells, and all of them are depended on the molecular mechanism of ER in BC cells.45,46\n\n\nEstrogen receptor and its classical mechanism of action\n\nEstrogen Receptor has a paramount role in BC cells, as described above. Hence it becomes the main target of the endocrine therapy such as ovarian blockade, SERM (Selective Estrogen Receptor Modulator, i.e., Tamoxifen), and SERD (Selective Estrogen Receptor Degrader, i.e., Fulvestran).10,22\n\nBeing a nuclear receptor family member, ER-α and ER-β are the two different types of Estrogen Receptors. In breast tissue, the ER-α has a dominant role. Meanwhile, ER-β is still considered controversial and has an unclear role.47 Another estrogen receptor type is the G-coupled Estrogen Receptor (GPER), which is paramount for estrogen molecular action via the membranous mechanism.48\n\nER-α coded by ESR-1 gene in chromosome 14, with an identical structure as other nuclear receptors, consists of 4 structural and functional domains. These domains are the amino-terminal domain (A/B domain), DNA binding domain/DBD, hinge region (D domain), and Ligand-Binding Domain/LBD.49\n\nAfter entering the cytoplasm of the cells, Estrogen will form a dimer form then bind to the C-domain of the ER at its 12th helix. Afterward, this complex (Estrogen dimer and ER) will be transported to the nucleus by D-domain.50\n\nSubsequently after entering the nucleus, DBD with the aid of co-activators will bind to Estrogen’s target genes that contain Estrogen Response Elements (EREs). The known co-activators are steroid receptor co-activator-1/SRC-1, SRC-2, SRC-3 (also known as AIB1/Amplified in Breast-Cancer 1). The binding of ER and EREs will activate the transcription of Estrogen’s target genes.4,51,52 This process is the so-called classic mechanism of ER molecular action, depicted in the animation below along with other mechanisms. This mechanism is the first known ER molecular action and has become the theoretical basis for applying the traditional endocrine therapy in luminal BC such as ovarian blockade, SERM, and SERD.10,24\n\n\nOther molecular actions of estrogen receptor\n\nThe estrogen receptor molecular actions are complicated and involve the intersecting apoptotic along with the survival pathways such as PI3K/Akt/mTOR, MAPK/ERK, resulting in the same similar target genes such as Cyclin-CDK, growth factor, and its activators.10,53 Currently, there are four known molecular mechanisms of ER actions: classical (genomic), non-classical, non-genomic (membranous), and ligand-independent (estrogen-independent). These four mechanisms are pictured in Figure 1.\n\nIn the non-classical mechanism (2nd mechanism in Figure 1), Estrogen could activate genes transcription that don’t contain EREs with help from tethering co-factors such as NF-kB (Nuclear Factor-Kappa Beta), activator protein 1 (AP-1), or specificity protein 1 (SP-1).4,10\n\nIn the membranous mechanism (3rd mechanism in animation), Estrogen will not be required to enter the nucleus to do the genomic action since ER receptors conduct all the inciting processes in the cell membrane. In the last mechanism, even Estrogen is not required to induce its target genes transcription (hence the name ligand-independent mechanism).4,10\n\nThese mechanisms will induce the exact effect on breast cancers cells: accentuating proliferative pathways and diminishing pro-apoptotic pathways. These non-classic, membranous, and particularly ligand-independent mechanisms result in cancer cells being more resistant to endocrine therapy. It is as if these estrogen receptors’ mechanism of action is being “hijacked” by the cells; the cancer cells are manipulating it to their benefit that is to replicate more and being less sensitive to apoptotic signals.5,10,22,48\n\nThe final result of these processes are constant activation of the estrogen receptor target genes although there were no more estrogen molecules available (i.e., due to ovarian blockade or inhibition by aromatase inhibitor), and although its receptor being blocked or degraded (i.e., due to inhibition by SERM/SERD).5,10,22,48\n\n\nProgesterone and progesterone receptor\n\nProgesterone is a steroid hormone produced by the corpus luteum in the human ovarium, which its primary duty is to prepare the female body for gestation. In breast epithelial cells, its role is indispensable in affecting ducto-alveolar changes in phases such as puberty, luteal phase (pre-menstrual period), pregnancy, and lactation.54\n\nCompared to Estrogen, the role of Progesterone in breast cancer cells, especially endocrine therapy, and its resistance is less distinctive and less studied. The cyclic level of Progesterone and its hundreds of active metabolites available in the female body are the main difficulties in testing this hormone. Furthermore, the target genes of ER and PR are overlapping, thus adding the complexity of this issue.55 But still, one cannot ignore the fact that the combination of Progesterone and Estrogen will add mitogenic effect to BC cells in the animal model, as epidemiological observations had shown.56–58\n\nPR was transcribed by three means. First, its transcription is induced by Estrogen as PGR (gene for encoding the PR) is one of the Estrogen target genes. Estrogen has been proven to be required in maintaining PR levels in breast and endometrium epithelial cells.59 Second, cancer cells could induce PR transcription mediated by Insulin Growth Factor-1 (IGF-1) and MAPK/ERK activity. Even more, at high Progestin concentration, these growth factors will be re-induced and thus will re-activate the ER-α phosphorylation in the ligand-independent mechanism of ER (review above animation), resulting in more PR transcription.60\n\nSome of the Progesterone target genes are also known to overlap with Estrogen target genes such as Cyclin-CDK, RANKL (Receptor activator of nuclear factor-kappa-Β ligand), and other growth factors. Hence these crosstalk mechanisms between ER and PR are crucial for breast cancer cells carcinogenesis and endocrine therapy resistance.5,60\n\nLike Estrogen, the action of Progesterone in cells is entirely dependent on its receptors, and it consists of both nuclear and membranous receptor types. There are two types of nuclear PR: PR-A and PR-B. Both nuclear receptors exist in breast epithelial cells in variable amounts and activity. Furthermore, it is unclear which nuclear receptor is more dominant in breast epithelial cells.55\n\nIdentical to ER, PR has N-terminal domain, Ligand Binding Domain /LB, Progesterone will bind the PR, DNA Binding Domain/DBD in which target genes contain Progesterone Receptor Elements (PREs) will bind.61\n\nAfter entering the cell’s cytoplasm, Progesterone will form a dimer, bind to PR in LBD, subsequently enter the nucleus, and bind to PREs with the aid of a co-factor. This process will activate the transcription of PR target genes. This mechanism is known as the PR action’s classical/direct genomic mechanism.55 Other mechanisms known are the non-classical/direct non-genomic and membranous mechanisms, depicted in Figure 2.16,55\n\nIn the indirect genomic mechanism, progesterone could activate genes that do not contain PREs as long there are tethering co-factors. In the membranous mechanism, PI3K/Akt/mTOR pathway and MAPK/ERK are also activated by progesterone. Therefore, it will accentuate the proliferative pathways and diminish the pro-apoptotic signals.16,55\n\n\np53 involvement in pathways activated in ER and PR molecular of actions\n\nThe complicated cellular signaling tightly regulates the cell cycle to maintain the regular cell proliferation rate and minimize error in DNA synthesis. In this cell cycle, abnormal cells with DNA error will be ceased in G1-S transition critical point.62\n\nEssentially, this critical G1-S transition point is determined by the interaction of Cyclin-D1& CDK4/6. In conditions without inhibition, this interaction will release E2F protein from its bond with Retinoblastoma Protein (RB Protein). The E2F protein will further trigger the cell to enter the S phase. Then consequently, abnormal cells with DNA will be duplicated.63\n\nThis mechanistic complex is one of the most often disrupted cellular signaling. It is found in endocrine therapy-resistant breast cancer cells, as Cyclin D1 (CCND1) and CDK4 become the target genes of Estrogen and Progesterone. Previously, Cyclin-CDK is still transcribed by the cancer cells, although the Estrogen production has been diminished and their receptors have been blocked.22,51,52 Relevantly, CDK 4/6 inhibitor has been approved in clinical guidelines as an adjunctive for endocrine therapy in Luminal BC, both pre- and post-menopausal patients.13,64\n\nIn normal cellular regulation, cells with abnormal DNA will be forced to enter G0 phase by the p21 protein, a protein transcribed and regulated by p53. This p21 protein will inhibit the CyclinD1-CDK4/6 complex, resulting in the cell entering the G0 phase and starting the DNA repairing process. This well-regulated system earned p53 the old nickname: “guardian of the genome”.65,66\n\nPI3K/Akt/mTOR pathway is a series of consecutive intracellular signaling that will activate proliferation and prevent apoptotic events. Genetic accumulation in this pathway and mutation of its inhibitor (PTEN/Phosphatase and TENsin homolog deleted on chromosome 10) are found about 70% from the whole BC population.12,67\n\nPI3K is an intracellular lipid kinase enzyme that will phosphorylate phosphatidylinositol molecule in the cell membrane, subsequently turning phosphatidylinositol-4,5-bisphosphate (PIP2) into phosphatidylinositol-3,4,5-trisphosphate (PIP3).67 Afterward, PIP3 will facilitate interaction between phosphoinositide-dependent kinase 1 (PDK-1) and Akt in the cell cytoplasm, resulting in a phosphorylated Akt. This phosphorylated Akt will activate Forkhead box O transcription factor (FoxO) that inhibits pro-apoptosis genes and also activates mechanistic targets of rapamycin (mTOR) complexes.12\n\nThe mTOR complexes are consist of 2 active forms: activated mTORC1 and mTORC2. mTORC1 will activate genes involved in carcinogeneses like protein synthesis, pro-survival genes, and cell growth. mTORC2 will specifically enhance phosphorylation, further causing Akt hyperactivation.12,67\n\nIn ER-α molecular actions, PI3K/Akt/mTOR will be activated in the non-classical, membranous, and ligand-independent mechanism.10,48,53,68 A likely, PI3K/Akt/mTOR will also be activated in PR molecular actions.16,55 Additionally, mTOR1 will activate S6K that will help to phosphorylate RE-α, further activating the functional domain of RE-α. Likewise, the Akt activates the NF-kB that functions as a co-factor in the non-classical and membranous mechanism of ER-α molecular actions.21\n\nIt is well known that PTEN, a classical tumor suppressor gene, will reverse PIP3 to PIP2; hence the subsequent Akt/mTOR activation will not occur. In the absence of PTEN, PI3K/Akt/mTOR pathway will be hyperactivated.69\n\nThe wild-type p53 protein will activate PTEN gene transcription. In cells with mutant p53, PTEN gene mRNA expression will be drastically reduced compared to cells with wild-type p53 status.70Another seminal finding by Jung, et al. 2018 in cell culture studies show cells with PTEN loss will cause PI3K/Akt/mTOR hyperactivation, causing mTORC1 and mTORC2 enhancement, and both will phosphorylate and activate wild-type p53 protein, which causes p21 transcription. p21 protein will further induce cells to premature senescence condition.71\n\nNuclear Factor-kappa Beta (NFKB) is a transcription factor family consisting of 5 subtypes: p50, p52, p65 (RelA), RelB, and c-Rel. With a vast target gene involved crucially in chronic inflammation and cellular proliferation, NF-kB is studied extensively in many cancers, including endocrine-resistant Luminal BC. In cell culture studies, treatment with NF-kB inhibitor will evoke the endocrine therapy sensitivity and toxicity; therefore, it has not been tested on humans.9\n\nNF-kB target genes considered instrumental in endocrine therapy resistance evolution are Cyclin D1, D2, D3 dan E, anti-apoptotic protein Bcl-2, MDM-2, and PDL-1 (Programmed Death Ligand-1).9,27,72 In ER molecular actions explained above; the NF-KB works as a co-factor in non-classical and membranous mechanisms so that Estrogen’s target genes are still transcribed although Estrogen has been blocked.9,72–76\n\nThe p53 and NF-kB have a negative association, for one cannot exist if the others are activated. The function is also contradictive; NF-kB will cause cell proliferation, anti-apoptotic, and enhance chronic inflammation, whereas p53 will regulate the cell cycle and trigger pro-apoptotic events when needed.72,77\n\nThe antagonistic mechanisms are various. Some studies noted wild-type p53 protein act as the direct promoter inhibitor of NF-kB target genes, therefore inhibiting the transcription of the NF-kB target genes. Others stated they compete with each other to get transcription co-factor 300. Wild-type p53 protein will also be known to inhibit the IKK enzyme (Inhibitor of Kappa Kinase, an enzyme to activate the active form of NF-kB). Without IKK, NF-kB couldn’t enter the nucleus and induce its target genes transcription.72,77,39\n\nTNF-α, the multi-functional mediator in inflammation and cells apoptosis, is also noted in Luminal BC for its role in enhancing cellular proliferation via NF-kB activation.78,79 The wild-type p53 was also recognized in turning off TNF-α induced NF-kB activation. This action is achieved by binding and blocking the work of Disabled homolog 2-interacting protein (DAB2IP), a protein that will activate TNF-α to trigger NF-kB activation.78\n\nSummary of all p53 works in ER-α and PR molecular actions could be seen in Figures 3 and 4.\n\n\nLimitation of p53 usage as a predictive biomarker in luminal breast cancer\n\nAlthough p53 is prominently correlated with poorer clinical features such as high proliferation index and higher grade and stadium, its usage in Luminal BC is still arguably limited.80 One possible cause is that the presence of ER seems to supress the p53 mutation itself.81 From epidemiological point of view, p53 mutation indeed is more frequently found in HER-2 enriched group and the Triple Negative BC group rather than Luminal BC. However, p53 mutation when found in Luminal BC is not without importance. In fact study by Lee, et al. 2013 from 7739 patients shown us that p53 mutation is correlated with higher proliferative index such as Ki-67 in Luminal A BC, and when combined together they effect long term survival of the patients.82\n\nSince it has been found 40 years ago, p53 protein has been published in thousands studies in numerous cancer either using protein detection or genetic testing. These factors along with versatility of p53 function in cells makes the test for p53 are widely known and readily available in most laboratories, therefore p53 is an ideal predictor to be chosen.38\n\np53 protein accumulation is easily detected by immunohistochemistry (IHC) as a surrogate marker of its mutation. However p53 immunohistochemistry testing in BC could present and correlates either with our without positive gene mutations tested, further affecting its capability as biomarker in BC.81 It is an obstacle that also frequently found in other cancers such as ovarian and gastric cancer.83,84\n\nNo clear cut off of p53 positivity in IHC assay also have been noted as the cause and affecting p53 usage as predictive biomarker in BC.85 Study by Kikuchi, et al. 2013 had tried to address this cut-off issue, found that when we set the cut-off of p53 immunoreactivity into ≥50% then it could be useful to predict clinical behaviour in Luminal Breast Cancer, especially Luminal B type (p<0.0001).86 This finding is also confirmed by another epidemiological study of 7226 patients by Abubakar, et al. in 2019.34\n\nFurthermore, p53 protein accumulation has been a limitation as a predictor due to many p53 protein isoforms formed within the tissue. These isoforms are many, with each was said to have its roles in molecular effect in cancer cells.31,81,87,88 This limitation has been countered with the suggestion of genetic testing such as PAM50 or Mammaprint that would replace the p53 protein accumulation testing. Although it has been deemed more accurate than IHC assay, the genetic testing is expensive and not readily available in most laboratories, becoming the deterrent factors for choosing this testing in clinical setting.89\n\n\nFuture perspective\n\nEndocrine therapy is a very beneficial therapy for Luminal BC patient. Its usage will decrease 15-years mortality rate up to 30-40%, consequently its resistance will pose the patients to dismal prognosis. As mentioned above, an established predictive biomarker will help clinicians to identify which patients will not have ET benefit in the first place, therefore their adjuvant therapy should be changed to other modalities to reduce recurrence and increase overall survival rate.2 In future perspective, predictive biomarker that could anticipate ET is certainly needed towards developing personalized and tailored therapy for Luminal BC patients.2,30\n\nDeveloping and planning studies to identify such biomarker is not easy since the complexities of the endocrine therapy resistance theories mentioned before. Estrogen metabolism in premenopausal and postmenopausal women are also different, hence the endocrine therapy given are different, therefore these group cannot be investigated together.5,28,48 With previous reasons mentioned, meticulously planned study embedded in RCT with carefully chosen patients and prospective analysis probably is best to identify such biomarker, explained very well in seminal study by Beelen, et al.4\n\nAdditionally, p53 mutation could occurred as early as pre-carcinogenesis period, in early stage of BC and in late/metastatic disease.20 Consequently it will be compulsory to test p53 mutation along the course of the disease and observe whether it correlate with ET resistance later.\n\np53 is also known to have particular effects in each type of breast cancer (luminal A/B, with or without HER2 positive status) due to BC heterogeneity.90 Several studies have been made to address this issue, and concluded that Luminal B breast cancer is the most probable BC group in which p53 mutation could be useful as predictive biomarker to predict ET resistance occurence.82,90 This fact is also supported by epidemiological data that showed p53 mutation was found in higher in Luminal B BC compared than Luminal A BC.33,34,91\n\nAnother interesting development is Neoadjuvant Endocrine Therapy (NET), which currently being studied in clinical trial, reportedly has advantages in downstaging and increasing Breast Conserving Therapy (BCT) success.92 In the future NET could reduce hospital stay for Luminal BC patients and outreach the undertreated patients group. When patients could not go to the hospital for various reasons, then endocrine therapy could provide more accessible and comfortable neo-adjuvant treatment than chemotherapy or radiotherapy.92 Therefore the needs to find such predictive biomarker become more pressing and indispensable, and we have to explore p53 mutation as plausible biomarker.\n\n\nConclusion\n\np53 is an important biomarker to be considered as an ideal candidate to anticipate ET resistance in the future since its role within pathways involved in the ER and PR molecular mechanisms is paramount and cannot be ignored. Its limitation as a predictor could be countered using proper genetic testing rather than protein marker. Well planned studies will be a prerequisite to conclude whether p53 truly useful as predictive biomarker for ET resistance in Luminal BC patients especially Luminal B group, with adequate period of observation.\n\n\nData availability\n\nNo data are associated with this article.\n\n\nAuthors’ contributions\n\nFH worked the Conceptualization, Data Curation, Formal Analysis, Funding Acquisition, Investigation, Methodology, Resources, Software, Visualization, Writing – Original Draft Preparation. YA involved in Conceptualization, Data Curation, Formal Analysis, Supervision, Validation, Writing – Review & Editing. SS involved in Project Administration, Resources, Software, Supervision, Writing – Review & Editing. FH wrote the draft of the article, YA and SS helped with final manuscript preparation. BH involved in Conceptualization, Project Administration, Software, Supervision, Validation, Writing – Review & Editing. All figures and animations are original to this manuscript, composed by FH and approved by YA, SS and BH. All authors read and approved the final manuscript.",
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PubMed Abstract | Publisher Full Text\n\nÁlvarez-Garcia V, Tawil Y, Wise HM, et al.: Mechanisms of PTEN loss in cancer: It’s all about diversity. Semin. Cancer Biol. 2019; 59(January): 66–79. Publisher Full Text\n\nStambolic V, MacPherson D, Sas D, et al.: Regulation of PTEN transcription by p53. Mol. Cell. 2001; 8(2): 317–325. Publisher Full Text\n\nJung SH, Hwang HJ, Kang D, et al.: mTOR kinase leads to PTEN-loss-induced cellular senescence by phosphorylating p53. Oncogene. 2019; 38(10): 1639–1650. Publisher Full Text\n\nPal S, Bhattacharjee A, Ali A, et al.: Chronic inflammation and cancer: Potential chemoprevention through nuclear factor kappa B and p53 mutual antagonism. J Inflamm. (United Kingdom). 2014; 11(1): 23. Publisher Full Text\n\nZhou Y, Eppenberger-Castori S, Eppenberger U, et al.: The NFκB pathway and endocrine-resistant breast cancer. Endocr. Relat. Cancer. 2005; 12(SUPPL. 1): 37–46.\n\nDolcet X, Llobet D, Pallares J, et al.: NF-kB in development and progression of human cancer. Virchows Arch. 2005; 446(5): 475–482. PubMed Abstract | Publisher Full Text\n\nNakshatri H, Bhat-Nakshatri P, Martin DA, et al.: Constitutive activation of NF-kappaB during progression of breast cancer to hormone-independent growth. Mol. Cell. Biol. 1997; 17(7): 3629–3639. PubMed Abstract | Publisher Full Text | Free Full Text\n\nWang X, Belguise K, O’Neill CF, et al.: RelB NF-κB Represses Estrogen Receptor α Expression via Induction of the Zinc Finger Protein Blimp1. Mol. Cell. Biol. 2009; 29(14): 3832–3844. PubMed Abstract | Publisher Full Text\n\nThomasova D, Mulay SR, Bruns H, et al.: p53-independent roles of MDM2 in NF-κB signaling: Implications for cancer therapy, wound healing, and autoimmune diseases. Neoplasia (United States). 2012; 14(12): 1097–1101. PubMed Abstract | Publisher Full Text\n\nRooks MG, Garrett W: p53 Mutations and Inflammation-Associated Cancer Are Linked through TNF Signaling. Mol. Cell. 2014; 56(5): 611–612. Publisher Full Text Reference Source\n\nMercogliano MF, Bruni S, Elizalde PV, et al.: Tumor Necrosis Factor α Blockade: An Opportunity to Tackle Breast Cancer. Front. Oncol. 2020; 10(April). PubMed Abstract | Publisher Full Text\n\nSadighi S, Zokaasadi M, Kasaeian A, et al.: The effect of immunohistochemically detected p53 accumulation in prognosis of breast cancer; a retrospective survey of outcome. PLoS One. 2017; 12(8): 1–10. Publisher Full Text\n\nCoates AS, Millar EKA, O’Toole SA, et al.: Prognostic interaction between expression of p53 and estrogen receptor in patients with node-negative breast cancer: Results from IBCSG Trials VIII and IX. Breast Cancer Res. 2012; 14(6): R143. PubMed Abstract | Publisher Full Text\n\nLee SK, Bae SY, Lee JH, et al.: Distinguishing Low-Risk Luminal A Breast Cancer Subtypes with Ki-67 and p53 Is More Predictive of Long-Term. Survival. 2015; 10: 1–14. Publisher Full Text\n\nKöbel M, Piskorz AM, Lee S, et al.: Optimized p53 immunohistochemistry is an accurate predictor of TP53 mutation in ovarian carcinoma. J. Pathol. Clin. Res. 2016; 2(4): 247–258. PubMed Abstract | Publisher Full Text\n\nFondevila C, Metges JP, Fuster J, et al.: p53 and VEGF expression are independent predictors of tumour recurrence and survival following curative resection of gastric cancer. Br. J. Cancer. 2004; 90(1): 206–215. PubMed Abstract | Publisher Full Text\n\nMilićević Z, Bajić V, Živković L, et al.: Identification of p53 and its isoforms in human breast carcinoma cells. Sci. World J. 2014; 2014: 1–10. PubMed Abstract | Publisher Full Text\n\nKikuchi S, Nishimura R, Osako T, et al.: Definition of p53 overexpression and its association with the clinicopathological features in luminal/HER2-negative breast cancer. Anticancer Res. 2013; 33(9): 3891–3897. PubMed Abstract\n\nBischof K, Knappskog S, Hjelle SM, et al.: Influence of p53 Isoform Expression on Survival in High-Grade Serous Ovarian Cancers. Sci. Rep. 2019; 9(1): 1–11.\n\nAvery-kiejda KA, Morten B, Wong-brown MW, et al.: The relative mRNA expression of p53 isoforms in breast cancer is associated with clinical features and outcome.2014; 35(3): 586–96.\n\nAlfarsi L, Johnston S, Liu DX, et al.: Current issues with luminal subtype classification in terms of prediction of benefit from endocrine therapy in early breast cancer. Histopathology. 2018; 73(4): 545–558. PubMed Abstract | Publisher Full Text\n\nAhn SH, Kim HJ, Han W, et al.: Breast Cancer Effect Modification of Hormonal Therapy by p53 Status in Invasive. Breast Cancer. 2013; 16(4): 386–394. PubMed Abstract | Publisher Full Text\n\nChuangsuwanich T, Pongpruttipan T, O-charoenrat P, et al.: Clinicopathologic features of breast carcinomas classified by biomarkers and correlation with microvessel density and VEGF expression: A study from Thailand. Asian Pac. J. Cancer Prev. 2014; 15(3): 1187–1192. PubMed Abstract | Publisher Full Text\n\nMadigan LI, Dinh P, Graham JD: Neoadjuvant endocrine therapy in locally advanced estrogen or progesterone receptor-positive breast cancer: Determining the optimal endocrine agent and treatment duration in postmenopausal women-a literature review and proposed guidelines. Breast Cancer Res. 2020; 22(1): 1–13. Publisher Full Text"
}
|
[
{
"id": "141770",
"date": "12 Jul 2022",
"name": "Didik Setyo Heriyanto",
"expertise": [
"Reviewer Expertise Airway",
"Cardiovascular",
"and Mediastinal Pathology",
"Gastrointestinal",
"Liver",
"and Gallbladder Pathology",
"General Pathology."
],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nSince 2002, p53-related breast cancer research has been conducted. Regularly, p53 is evaluated for diagnostic and prognostic purposes. In contrast to other solid tumors, breast cancer has a lower incidence of p53 gene mutations. The reviewer suggests that the author also provide updates on recently studied potential biomarkers, particularly those associated with breast cancer. Given that the TP53 mutation is associated with a more aggressive disease and a lower overall survival rate, it is also crucial to identify a potential biomarker that can be tested in the early stages of the disease1.\nThis study also states that p53 gene mutation is one of the most common genetic alterations in breast cancer, but its incidence is only 30–35% in Luminal B BC , compared to 70% in Her-2/neu overexpression and 80% in TNBC2. This is also stated as a limitation in the study. The reviewer suggest that the study could also provide additional insight into the recent roles of p53 gene mutation applications as biomarkers in Her-2/neu overexpression and the TNBC group, in which both have higher incidence3.\nIt is possible for endocrine resistance to develop during the endocrine therapy or at a later time. While it is possible that the p53 gene mutation is suppressed by the presence of ER, this also means that individuals who are more susceptible to endocrine therapy have a greater chance of doing so naturally. In addition, the reviewer emphasize the importance of determining whether endocrine therapies that interact with ER, such as SERM and SERD, would also affect its suppressive effect on p53 mutation4,5.\nIn conclusion, p53 has the potential to become a biomarker for predicting ET resistance in the future, as its participation in the pathways involving the ER and PR molecular mechanisms is essential and cannot be ignored. The author has elaborated on the limitations of the predictability tool. The reviewer concur that additional research is necessary to determine whether p53 is effective as a prognostic biomarker for ET resistance in Luminal BC patients.\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": "8517",
"date": "20 Jul 2022",
"name": "Freda Halim",
"role": "Author Response",
"response": "Dear reviewer, thank you for your comment regarding our article. Indeed we believe p53 is an important marker to be checked in luminal breast cancer because it plays many seminal roles in endocrine resistance pathways. We are delighted that your comment on our work supports this."
}
]
},
{
"id": "152460",
"date": "17 Oct 2022",
"name": "Norbert Nass",
"expertise": [
"Reviewer Expertise Cell Biology",
"Tamoxifen resistance in breast cancer",
"protein biosynthesis"
],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nIn this review the authors summarize our current knowledge on a possible function of p53 in the occurrence of resistance towards endocrine therapy in breast cancer. The main hypothesis is that p53 might regulate estrogen receptor expression, abundance and signalling. Deregulation or mutation of p53 then causes resistance because the estrogen receptor is a major mediator of endocrine therapy. Therefore p53 should be considered in biomarker studies for anti-endocrine resistance. This is an interesting review as p53 mutations and abundance have not conclusively been investigated as a potential biomarker for anti-endocrine resistance. It is also carefully written and illustrated.\nBesides the mechanisms discussed in this manuscript, there is cumulating evidence that the cancer microenvironment as well as non-coding RNAs are also involved in the development of endocrine therapy resistance. I suggest, this should be mentioned here.\nThe authors also state that \"Estrogen will form a dimer form, then bind to…”. I am not sure if this is true. In my current view, one estrogen molecule binds to one receptor binding domain. Then the estrogen receptor dissociates from e.g. heat shock proteins and forms dimers. Please clarify!\nLater, the authors state “The final result of these processes are constant activation of the estrogen receptor target genes”. I think this not the whole story. Many gene expression studies on e.g. tamoxifen or fulvestrant adapted cell lines show that many apparently estrogen-unrelated genes are also regulated.\nI think it would be worthwhile mentioning that PGR and ESR1 are usually co-expressed and only a few cases are described that express PGR, but not ESR1.\n\nIs the topic of the review discussed comprehensively in the context of the current literature? Yes\n\nAre all factual statements correct and adequately supported by citations? Yes\n\nIs the review written in accessible language? Yes\n\nAre the conclusions drawn appropriate in the context of the current research literature? Partly",
"responses": [
{
"c_id": "9009",
"date": "30 Nov 2022",
"name": "Freda Halim",
"role": "Author Response",
"response": "\"In this review, the authors summarize our current knowledge on a possible function of p53 in the occurrence of resistance towards endocrine therapy in breast cancer. The main hypothesis is that p53 might regulate estrogen receptor expression, abundance, and signaling. Deregulation or mutation of p53 causes resistance because the estrogen receptor is a major mediator of endocrine therapy. Therefore p53 should be considered in biomarker studies for anti-endocrine resistance. This is an interesting review as p53 mutations and abundance have not conclusively been investigated as a potential biomarker for anti-endocrine resistance. It is also carefully written and illustrated.\" Dear Reviewer, we are grateful for your gracious review of our article, and we attempt to address several issues raised in the comment. \"Besides the mechanisms discussed in this manuscript, there is cumulating evidence that the cancer microenvironment, as well as non-coding RNAs, are also involved in the development of endocrine therapy resistance. I suggest this should be mentioned here.\" Answer: We have already added several postulations about the tumor microenvironment's role in ET resistance and non-coding RNAs. \"The authors also state that \"Estrogen will form a dimer form, then bind to…\". I am not sure if this is true. Currently, one estrogen molecule binds to one receptor-binding domain. Then the estrogen receptor dissociates from, e.g., heat shock proteins and forms dimers. Please clarify!\" Answer: we acknowledge this error and have already edited the paragraph. \"Later, the authors state, \"The final result of these processes is constant activation of the estrogen receptor target genes.\" I think this is not the whole story. Many gene expression studies on, e.g., tamoxifen or fulvestrant-adapted cell lines show that many apparently estrogen-unrelated genes are also regulated.\" Answer: We acknowledged these facts and edited the sentence. We already mentioned that several mechanisms of estrogen-unrelated genes are also regulated in the tamoxifen and fulvestrant-adapted cell lines. \"I think it would be worthwhile mentioning that PGR and ESR1 are usually co-expressed, and only a few cases are described that express PGR but not ESR1.\" Answer: We agreed to this comment and already mentioned it in our review, with cited literature."
}
]
}
] | 1
|
https://f1000research.com/articles/11-330
|
https://f1000research.com/articles/11-245/v1
|
28 Feb 22
|
{
"type": "Brief Report",
"title": "Negative E-cadherin expression on bone marrow myeloma cell membranes is associated with extramedullary disease",
"authors": [
"Maki Hirao",
"Kohei Yamazaki",
"Kentaro Watanabe",
"Kiyoshi Mukai",
"Shigemichi Hirose",
"Makoto Osada",
"Yuiko Tsukada",
"Hisako Kunieda",
"Ryunosuke Denda",
"Takahide Kikuchi",
"Hiroki Sugimori",
"Shinichiro Okamoto",
"Yutaka Hattori",
"Kohei Yamazaki",
"Kentaro Watanabe",
"Kiyoshi Mukai",
"Shigemichi Hirose",
"Makoto Osada",
"Yuiko Tsukada",
"Hisako Kunieda",
"Ryunosuke Denda",
"Takahide Kikuchi",
"Hiroki Sugimori",
"Shinichiro Okamoto"
],
"abstract": "Background: The loss of E-cadherin expression and the induction of N-cadherin are known as hallmarks of the epithelial-to-mesenchymal transition, an essential initial step in the process of metastasis in solid tumors. Although several studies have reported expressions of these cadherins in patients with multiple myeloma (MM), their clinical significance is unknown as MM cells are non-epithelial. Methods: In this study, we examined the expression of E- and N-cadherins by immunohistochemistry using bone marrow (BM) biopsy specimens from 31 newly diagnosed MM patients and in subsequent biopsy specimens from six of these. Results: Negative E-cadherin expression on BM myeloma cell membranes was significantly associated with the presence of soft-tissue masses arising from bone lesions and breaking through the cortical bone, referred to as extramedullary disease (EMD). Conclusions: Given the aggressive nature of EMD, our study suggests that screening for E-cadherin using BM immunohistochemistry is one measure that could predict the development of EMD in patients with MM.",
"keywords": [
"E-cadherin",
"extramedullary disease",
"multiple myeloma",
"immunohistochemistry"
],
"content": "Introduction\n\nThe loss of E-cadherin expression and the subsequent induction of N-cadherin are known as hallmarks of the epithelial-to-mesenchymal transition, an essential initial step in the process of metastasis in solid tumors.1 Although multiple myeloma (MM) cells are non-epithelial cells, Roccaro et al. and Azab et al.2,3 suggested that reduced E-cadherin expression in MM cells can promote extramedullary disease (EMD) in vitro and in an animal model, partly due to epithelial-to-mesenchymal-transition-like features. EMD is defined as the presence of an MM-cell tumor outside the bone marrow (BM), either in the form of a soft-tissue mass spreading contiguously from the bone and breaking out of the cortical bone (osseous) or arising in an isolated organ not contiguous with bone lesions (extraosseous).4 Epithelial-to-mesenchymal transition-like features in MM patients can be used as biomarkers for EMD; however, the clinical significance of E- and N-cadherin expressions, especially in BM specimens of patients with MM, is still largely unknown and thus needs to be elucidated.\n\nIn this article, we examined E- and N-cadherin expression on MM cells obtained from patients using immunohistochemistry. We tried to clarify whether the expression of these cadherins is associated with the presence of EMD and other clinical parameters.\n\n\nMethods\n\nThis study was approved by the ethics committee of Saiseikai Central Hospital (Rin 28-66) and Keio University Faculty of Pharmacy (190605-3). Written informed consent for publication of the patients’ details and their images was obtained from the patients. This retrospective study included 89 consecutive patients (49 men and 40 women) between the ages of 36-94 years old (mean: 70 ± 13 years) between January 2009 and December 2016 who were newly diagnosed as having MM at Saiseikai Central Hospital. All eligible study patients with newly diagnosed MM did not receive any therapy at the time of BM biopsy. Inclusion criteria were provision of full data available on EMD involvement (yes or no) at time of diagnosis, its location, and the number of EMDs. After the exclusion of cases who were lost at follow-up or lacked indispensable medical documents, 31 patients (18 men and 13 women) between the ages of 46-88 years old (mean: 67 ± 11 years) were selected for this study. A total of 31 BM samples were collected at diagnosis and five BM samples and one surgically removed extraosseous EMD sample were collected during disease progression. The median observation period was 46 months (range 3-93).\n\nDisease stage for each patient was based on the International Staging System.16 Serum concentrations of albumin, β2 microglobulin, calcium, creatinine, lactate dehydrogenase and hemoglobin were measured as a part of routine medical care.\n\nImmunohistochemistry for E- and N-cadherin was performed on BM and EMD biopsy specimen in 31 patients. Tissue sections were cut from formalin-fixed, paraffin-embedded blocks containing MM cells and were processed for immunohistochemistry using an automated staining system (BOND-III, Leica Microsystems, Wetzlar, Germany). For assessment of E- and N-cadherin and CD138 expressions, the E-cadherin rabbit monoclonal antibody (Cell Signaling Technology Cat# 3195, RRID:AB_2291471), N-cadherin mouse monoclonal antibody (Abcam Cat# ab98952, RRID:AB_10696943) and CD138 mouse monoclonal antibody (Agilent Cat# M7228, RRID:AB_2254116) were applied at 1:600, 1:4,000 and 1:50 dilutions, respectively, followed by detection using the BOND Polymer Refine Detection kit (Leica Biosystems Cat# DS9800, RRID:AB_2891238). Antigen retrieval was performed with BOND Epitope Retrieval Solution 2 (EDTA, pH=9.0). Positive staining of MM cells was independently evaluated by two pathologists, K.M. and S.H., without any clinical information (Cohen’s κ score 1.0). At a magnification of 200, one field with the highest cadherin positive myeloma cell count on each slide was selected. The percentage of E- or N-cadherin-positive MM cells was calculated using the equation E- or N-cadherin-positive MM cells/total BM nucleated cells, regardless of the number of hematopoietic cells. A score >0.5% was used to determine positivity (yes vs. no) (Figure 1). The percentage of BM plasma cells was calculated using the equation CD138-positive cells/total BM nucleated cells, regardless of the number of hematopoietic cells.\n\n(A) BM biopsy with MM cell infiltrate. Hematoxylin and eosin stain. Consecutive sections of BM biopsies were stained with antibodies against (B) CD138 and (C) E-cadherin. Note that E-cadherin was clearly stained in the membrane of MM cells. MM, multiple myeloma; BM, bone marrow.\n\nThe presence of EMD was evaluated by well-trained radiologists. 18F-fluorodeoxyglucose positron emission tomography (PET)/computed tomography (CT) was used in 16/31 cases (51.6%) in total at diagnosis. For the remaining 15 cases, only CT, magnetic resonance imaging (MRI) or both was used in five, four, and six cases, respectively. For the five patients from whom second BM samples were taken during progression, PET/CT, only CT, and both CT and MRI were used in two, two and one cases, respectively. For the patient whose EMD sample was taken during progression, CT was used.\n\nStatistical analysis was performed with SAS 9.4 software (SAS institute, Cary, NC, USA; http://www.sas.com; Statistical Analysis System, RRID:SCR_008567). To compare clinical parameters between cadherin-positive and -negative MM patients, for continuous variables (i.e., age, serum concentrations of albumin, β2 microglobulin, calcium, creatinine, lactate dehydrogenase, hemoglobin, and BM plasma cell %), we performed pooled t-test for equal variances using the t-test analysis. Furthermore, for categorical variables we used the FREQ procedure to perform Fisher’s exact test for the data frequency table (i.e., sex and presence of EMD) because of the large number of cells with count less than five, and chi-square test for monoclonal protein subtypes and the International Staging System. The Kaplan–Meier method was used to analyze the survival probability distribution of the groups accounting for the E-cadherin expression. The statistical significance level was given at p <0.05 (two-sided).\n\n\nResults\n\nFigure 1 shows typical E-cadherin positivity by immunohistochemistry in a patient with MM. Table 115 depicts the results of E-cadherin expression in conjunction with various clinical parameters at diagnosis. E-cadherin expression was positive in 25 (80.6%) of the initial 31 BM samples. At initial screening, EMD was found in 14 (45.2%) cases. Four additional patients developed EMD on follow-up from 17 patients who had no EMD at diagnosis. Negative E-cadherin expression at diagnosis (found in six patients) was significantly associated with the presence of EMD (p=0.0041). At diagnosis, all 14 (100%) of the patients with EMD had osseous lesions; none (0%) had extraosseous lesions (Table 2). No association was found between N-cadherin expression and the presence of EMD.\n\n* Statistically significant.\n\n# Fisher’s exact test.\n\n♭ chi-square test.\n\nIn five (patients #25, 28, 29, 30 and 31) of the 31 newly diagnosed patients, four of whom (#25, 28, 29 and 31) were initially positive for E-cadherin and three of whom (#25, 28 and 30) had EMD, we were able to investigate E-cadherin status in an additional BM sample, later in disease progression. In two patients (#25 and 28), the second BM sample became negative for E-cadherin after the first had been positive; in both cases, new EMDs (two additional osseous EMDs in #25 and pleural and chest wall EMDs in #28) were present at the time of the second sample. The two (patients #29 and 31) who were positive for E-cadherin at both biopsies did not show EMD, but patient #30 who was negative for E-cadherin at both biopsies showed osseous EMD throughout the course of the disease, with an increase from one to more than 20. Furthermore, in patient #24, who developed testicular EMD during disease progression, E-cadherin expression was negative both in the BM sample at diagnosis and in the EMD sample at the time of disease progression.\n\nPatients with E-cadherin-positive cells had significantly higher serum albumin concentrations (p=0.0221), and hemoglobin values (p=0.0268) (Table 1). Figure 2 shows the overall survival (OS) probability in all enrolled patients. The median OS values of positive and negative E-cadherin patients were 51.5 months (range 3-93) and 25.8 months (range 6-62), respectively (Log-rank p=0.0644, Wilcoxon p=0.0682, -2Log (LR) p=0.0443). By contrast, N-cadherin expression was not significantly associated with any clinical parameters.\n\nOS stratified by the E-cadherin negativity of MM cells. A statistical significance in OS was found between the E-cadherin-positive and -negative groups (Log-rank p=0.0644, Wilcoxon p=0.0682, -2Log (LR) p=0.0443). Hashmarks indicate surviving patients at last visit. OS, overall survival; MM, multiple myeloma.\n\n\nDiscussion\n\nOur present report suggests that the loss of E-cadherin expression in MM cell membranes in BM is involved in the pathogenesis of osseous EMD. Negative E-cadherin expression was significantly associated with the presence of osseous EMD (p=0.0041) (Table 1). Few studies so far have specifically addressed whether epithelial-to-mesenchymal-transition-like features predict EMD. One experimental study demonstrated that cell lines overexpressing CXCR4 showed decreased E-cadherin.2 This study demonstrated progression to EMD in a mouse model subjected to the injection of this cell line. One clinical study provided evidence that the expression frequency of Twist1, which suppresses E-cadherin, in EMD samples from MM patients, is higher than that in BM samples from these patients or MM patients without EMD.5 In another study, the process of EMD formation has been associated with the upregulation of CXCR4 in EMD specimens of patients with MM.6 Notably, our study is the first to examine the E-cadherin expression of MM cells using BM biopsy specimens from patients.\n\nIn agreement with the hypothesis that loss of E-cadherin indicates development of EMD, in two out of five patients with follow-up BM samples (#25 and #28), E-cadherin was found to be positive at the time of diagnosis but negative at the time of disease progression. Interestingly, at progression, the number of osseous EMDs increased in patient #25 and new extraosseous pleural and chest wall EMDs developed in patient #28. In other cases, E-cadherin positivity (#29 and 31) or negativity (#30) was observed in both the first sample and that taken later in the course of the disease; one of these (#30) had EMD. These findings are consistent with the association of negative E-cadherin expression with the higher incidence of EMD, which seemed to be related to the shorter OS. In another patient with sequential samples, #24, a testicular EMD sample during disease progression was investigated. E-cadherin was found to be unexpressed in both the BM at diagnosis and in the EMD at the time of disease progression, possibly indicating the outgrowth of clones without E-cadherin during disease evolution. Taken together, these results suggest that clones without E-cadherin expression in the BM are \"fit\" under pressure and are assumed to be able to produce specific tropic growth to the EMD site.\n\nThe incidence of osseous EMD in this study was 45.2%. In the literature, osseous EMD is only present in up to 32.5% of newly diagnosed MM cases.7 The higher frequency in our patients is probably explained by the fact that our cohort was enriched by patients using PET/CT (51.6%) at initial staging. Indeed, PET/CT has shown superior potential to regular CT in detecting osseous EMD.8\n\nAnother objective of the current study was to investigate whether expression of E-cadherin was associated with clinicopathological parameters, and particularly OS, in these patients. In addition to the significant difference in OS (25.8 vs. 51.5 months; p=0.0443), it is noteworthy that negative E-cadherin expression in MM patients in this study was associated with lower serum albumin and hemoglobin levels, suggesting that the loss of this molecule is either a deteriorating factor or a marker for disease severity and shorter OS. Several pieces of circumstantial evidence support the loss of E-cadherin being closely associated with the progression of MM. A study using the MM mouse model showed that decreased E-cadherin expression was associated with tumor progression.3 In a clinical study using DNA-methylation analysis, the E-cadherin gene was shown to be silenced with increased frequency in MM than in patients with monoclonal gammopathies .9 Another clinical study using immunohistochemistry in spinal plasmacytoma, including 28 MMs with osseous EMD and 13 solitary plasmacytomas of bone, showed that patients with negative E-cadherin expression had a significantly shorter OS than those with positive (26.0 vs. 48.7 months; p <0.05).10 Although the immunohistochemistry samples were taken from surgically removed spinal masses in their study, their finding was in good accordance with our results in which the immunohistochemistry samples were taken from BM. However, the role of E-cadherin in disease progression and severity is still a matter for debate because the research results are not always consistent.11 As to the relationship between N-cadherin and patient prognosis, Vandyke et al.12 revealed an inverse relationship between circulating N-cadherin and patient OS. Contrarily, in our study, no statistically significant relationship was observed between N-cadherin expression in MM cells and patient prognosis.\n\nThere are some limitations in this study. The relatively small number of patients and incomplete data on cytogenetics and molecular biology make it difficult for us to assess the direct impact of E-cadherin expression of MM cells on OS. Therefore, further studies with a larger sample size and more consistent follow-up are required to confirm the association of negative E-cadherin expression with poor survival in MM patients with EMD. From the therapeutic viewpoint, preventive strategies to inhibit EMD have been proposed.13,14 Given the aggressive nature of EMD, biomarkers for patients at risk of EMD will help develop precision medicine for patients with MM.\n\nTaken together, our results suggest that further investigation into the usefulness of E-cadherin expression in simple and routine BM as a biomarker for EMD is warranted.\n\n\nData availability\n\nDryad: Negative E-cadherin expression on bone marrow myeloma cell membranes is associated with extramedullary disease. https://doi.org/10.5061/dryad.ns1rn8pvn.15\n\nData are available under the terms of the Creative Commons Zero “No rights reserved” data waiver (CC0 1.0 Public domain dedication).",
"appendix": "Acknowledgements\n\nWe thank Satoru Kuriyama (MD, PhD), Otoya Miho (MD, PhD), Maiko Matsushita (MD, PhD) and Daiju Ichikawa (PhD) for their critical reading of the manuscript; and Megumi Ishida and Asami Kamoya for their help in the acquisition of data.\n\n\nReferences\n\nLoh C-Y, Chai JY, Tang TF, et al.: The E-Cadherin and N-Cadherin Switch in Epithelial-to-Mesenchymal Transition: Signaling, Therapeutic Implications, and Challenges. Cells. 2019; 8(10): 1118. PubMed Abstract | Publisher Full Text\n\nRoccaro Aldo M, Mishima Y, Sacco A, et al.: CXCR4 Regulates Extra-Medullary Myeloma through Epithelial-Mesenchymal-Transition-like Transcriptional Activation. Cell Reports. 2015 2015/07/28; 12(4): 622–635. PubMed Abstract | Publisher Full Text\n\nAzab AK, Hu J, Quang P, et al.: Hypoxia promotes dissemination of multiple myeloma through acquisition of epithelial to mesenchymal transition-like features. Blood. 2012 Jun 14; 119(24): 5782–594. PubMed Abstract | Publisher Full Text | Free Full Text\n\nBansal R, Rakshit S, Kumar S: Extramedullary disease in multiple myeloma. Blood Cancer J. 2021 Sep 29; 11(9): 161. PubMed Abstract | Publisher Full Text | Free Full Text\n\nYang JZ, Lian WG, Sun LX, et al.: High nuclear expression of Twist1 in the skeletal extramedullary disease of myeloma patients predicts inferior survival. Pathol Res Pract. 2016 Mar; 212(3): 210–216. PubMed Abstract | Publisher Full Text\n\nWeinstock M, Aljawai Y, Morgan EA, et al.: Incidence and clinical features of extramedullary multiple myeloma in patients who underwent stem cell transplantation. Br J Haematol. 2015; 169(6): 851–858. PubMed Abstract | Publisher Full Text\n\nVarga C, Xie W, Laubach J, et al.: Development of extramedullary myeloma in the era of novel agents: no evidence of increased risk with lenalidomide-bortezomib combinations. Br J Haematol. 2015 Jun; 169(6): 843–850. PubMed Abstract | Publisher Full Text\n\nTian C, Wang L, Wu L, et al.: Clinical characteristics and prognosis of multiple myeloma with bone-related extramedullary disease at diagnosis. Biosci Rep. 2018 Jun 29; 38(3). PubMed Abstract | Publisher Full Text | Free Full Text\n\nSeidl S, Ackermann J, Kaufmann H, et al.: DNA-methylation analysis identifies the E-cadherin gene as a potential marker of disease progression in patients with monoclonal gammopathies. Cancer. 2004 Jun 15; 100(12): 2598–606. PubMed Abstract | Publisher Full Text\n\nWang L, Liu H, He J, et al.: E-cadherin expression and its clinical significance in 41 cases of spinal plasma cell myeloma. Journal of Medical Colleges of PLA. 2009 2009/02/01; 24(1): 56–62. Publisher Full Text\n\nBi E, Li R, Bover LC, et al.: E-cadherin expression on multiple myeloma cells activates tumor-promoting properties in plasmacytoid DCs. J Clin Invest. 2018 Nov 1; 128(11): 4821–4831. PubMed Abstract | Publisher Full Text | Free Full Text\n\nVandyke K, Chow AW, Williams SA, et al.: Circulating N-cadherin levels are a negative prognostic indicator in patients with multiple myeloma. Br J Haematol. 2013 May; 161(4): 499–507. PubMed Abstract | Publisher Full Text\n\nUsmani SZ, Weiss BM, Plesner T, et al.: Clinical efficacy of daratumumab monotherapy in patients with heavily pretreated relapsed or refractory multiple myeloma. Blood. 2016 Jul 7; 128(1): 37–44. PubMed Abstract | Publisher Full Text | Free Full Text\n\nGagelmann N, Ayuk F, Atanackovic D, et al.: B cell maturation antigen-specific chimeric antigen receptor T cells for relapsed or refractory multiple myeloma: A meta-analysis. Eur J Haematol. 2020 Apr; 104(4): 318–327. PubMed Abstract | Publisher Full Text\n\nHirao M: Negative E-cadherin expression on bone marrow myeloma cell membranes is associated with extramedullary disease, Dryad, Dataset.2022. Publisher Full Text\n\nGreipp PR, San Miguel J, Durie BG, et al.: International staging system for multiple myeloma. J Clin Oncol. 2005 May 20; 23(15): 3412–3420. Erratum in: J Clin Oncol. 2005 Sep 1;23(25):6281. Harousseau, Jean-Luc [corrected to Avet-Loiseau, Herve].PubMed Abstract | Publisher Full Text"
}
|
[
{
"id": "126959",
"date": "19 Apr 2022",
"name": "Antonio Bedalov",
"expertise": [
"Reviewer Expertise Cancer biology",
"hematologic malignancies",
"epigenetics",
"DNA replication"
],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nEpithelial to mesenchymal transition (EMT) and resulting alterations in cell adhesion, cell polarity, and cytoskeleton remodeling have been associated with the acquisition of invasive and metastatic properties of cancer cells. EMT is orchestrated by a well-characterized set of transcription factors (e.g. SNAIL, TWIST) which change the expression of several adhesion proteins, including downregulation of E-cadherin and upregulation of N-cadherin. Even though the hematopoietic system is mesoderm-derived, an EMT-like transcriptional cascade has been described in hematologic cancers, including multiple myeloma (MM) as shown in a mouse model of MM progression 1.\nA major finding of the study is that the lack of expression of E-cadherin in diagnostic bone marrow biopsy of 31 multiple myeloma patients is associated with the presence of extramedullary disease. All 6 patients that lack E-cadherin expression exhibit extramedullary disease (EMD) 6/6=100%), while among the 25 patients that express E-cadherin EMD is found at diagnosis in only 8 patients (8/25=32%). Even though the sample size is small, this association is significant and strong, which strengthens the argument that the EMT might be involved in the acquisition of invasive phenotype in MM. The authors have also observed that the absence of cadherin E expression is associated with reduced survival, however, it is presently not clear whether the lack of cadherin E expression was an independent prognostic marker or just a marker of more severe/advanced disease, given that other markers of disease severity such as reduced albumin and hemoglobin level are also associated with the absence of cadherin E expression. While limitations of the studies are mentioned. I believe that additional points that should be brought up in the discussion are:\nLess than half of the patients with EMD exhibit loss of cadherin E expression at diagnosis (does this reflect MM heterogeneity, sampling error, or do the authors believe that invasive phenotype occurs in EMT-independent fashion);\n\nDoes the prognosis of the patients with EMD but without loss of cadherin E differ from that in patients with EMD and loss of cadherin E;\n\nAre there patients for which the biopsy of both EMD and bone marrow biopsy have been obtained, and if so, what was the concordance in cadherin E expression between the two?\nIf unable to address these points directly because of the small sample size, this limitation should be acknowledged and discussed.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate? Yes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes",
"responses": [
{
"c_id": "8208",
"date": "10 May 2022",
"name": "Maki Hirao",
"role": "Author Response",
"response": "Thank you for your useful comments and suggestions. The reviewer has asked for a description of why loss of cadherin E expression at diagnosis was observed only in the 43% of the EMD observed cases (6 out of 14). MM is a clinically as well as biologically heterogeneous disease. Thus, we think loss of E-cadherin expression is not only the mechanism for EMD formation, but diverse molecular mechanisms exist for it. Currently, the small number of patients has only been used in assessment of the direct impact of E-cadherin expression of MM cells on OS, and we are unable to rule out the possibility of sampling errors. Therefore, our results require further studies with a larger sample size and more consistent follow-up. We will include this explanation in the discussion of the revised manuscript. Additional data analysis was performed to address potential roles of E-cadherin. Among the 14 patients with EMD, no correlation was found between E-cadherin expression and the clinical outcome. The median OS values of positive and negative E-cadherin patients were 34.9 months (range 5-87) and 25.8 months (range 6-62), respectively (Log-rank p=0.8459, Wilcoxon p=0.6507, -2Log (LR) p=0.6663). Further studies should be addressed to investigate the potential role of cadherin E in EMD patients for prognostic prediction. We note that the reviewer is concerned about the correlation in cadherin E expression between BM staining and EMD lesion. To address this issue, we have validated the concordance in cadherins E expression between the testicular EMD and BM biopsies only in patient #24. E-cadherin was found to be unexpressed in both the BM and the EMD. The findings are described on line 27 to 28 of page 4 and line 13 to 16 of page 6 of the manuscript. We are planning to carry out further studies on the EMD biopsies."
}
]
},
{
"id": "134806",
"date": "18 May 2022",
"name": "Makoto Sasaki",
"expertise": [
"Reviewer Expertise Multiple Myeloma",
"Clinical Trials"
],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe authors analyzed E-cadherin expression of clonal plasma cells in bone marrow using the immunohistochemistry staining and revealed the significant association between lack of expression of E-cadherin and osseous extramedullary disease (EMD) in patients with multiple myeloma (MM). Furthermore, they exposed the association of negative expression of E-cadherin with poor survival in patients with MM. Although the sample size is relatively small, this study provides essential information for physician-scientists in the hematological field.\nThere is some evidence of the association between CXCR4 and EMD. In some mouse myeloma models, low expression of CXCR4 is associated with progression of EMD1, and CXCR4 overexpression enhances epithelial-to-mesenchymal transition (EMT) like features with upregulation of Snail, Slug, and Twist, and downregulation of E-cadherin2. It would be more informative if the authors could evaluate and discuss the expression of CXCR4 in their patients.\n\nIn this report, EMD was observed in 14(45%) of 31 cases at diagnosis. Although, the incidence of EMD in myeloma is around 5% at diagnosis and 20-30% in relapsed/refractory patients 3,4.What is the cause of the extremely high prevalence of EMD in this study group?\n\nIt is better to add each patient's type of light chain in table 2. In the previous report cited by the authors, the expression of E-cadherin had a strong association with the type of light chain5\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate? Yes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes",
"responses": [
{
"c_id": "9139",
"date": "04 Jan 2023",
"name": "Maki Hirao",
"role": "Author Response",
"response": "The reviewer has requested that we provide information concerning the expression of CXCR4 in this study participants. We did not look at the expression of CXCR4 in this study. We acknowledge the need to consider the expression of CXCR4, however, it is currently not feasible. We think that high CXCR4 expression can contribute to the pathogenesis of EMD by loss of E-cadherin. The reviewer has commented on the high prevalence of EMD in this study. The reason for this was that most participants were intensively examined for EMD using PET/CT. We noted that Tian et al. (2018) used the same methods of collecting data in a study with similar prevalence of EMD. We accept, however, that the exact reason for high prevalence of EMD is still unclear. Thank you for drawing out attention to the type of light chain. If we understand correctly, the reviewer would like to know about the relationship between the type of light chain and the expression of E-cadherin. The reference should be Wang et al. (2009), instead of Bi et al. (2018), and the Table number should be 1, instead of 2. Wang et al. (2009) found that the expression of E-cadherin in the λ chain subgroup was significantly lower than that in the κ chain group (P < 0.05). Thus, additional data analysis was performed to address the relationship in our study participants. The results are reported in the revised version of Table 1. In our study, the correlation was not statistically significant (p=0.1719)."
}
]
}
] | 1
|
https://f1000research.com/articles/11-245
|
https://f1000research.com/articles/11-1007/v1
|
07 Sep 22
|
{
"type": "Method Article",
"title": "Development of liquid culture media mimicking the conditions of sinuses and lungs in cystic fibrosis and health",
"authors": [
"Dilem Ruhluel",
"Siobhan O'Brien",
"Joanne L Fothergill",
"Daniel R Neill",
"Siobhan O'Brien"
],
"abstract": "The respiratory tract is a compartmentalised and heterogenous environment. The nasopharynx and sinuses of the upper airways have distinct properties from the lungs and these differences may shape bacterial adaptation and evolution. Upper airway niches act as early colonisation sites for respiratory bacterial pathogens, including those, such as Pseudomonas aeruginosa, that can go on to establish chronic infection of the lungs in people with cystic fibrosis (CF). Despite the importance of upper airway environments in facilitating early adaptation to host environments, currently available in vitro models for study of respiratory infection in CF focus exclusively on the lungs. Furthermore, animal models, widely used to bridge the gap between in vitro systems and the clinical scenario, do not allow the upper and lower airways to be studied in isolation. We have developed a suite of culture media reproducing key features of the upper and lower airways, for the study of bacterial adaptation and evolution in different respiratory environments. For both upper and lower airway-mimicking media, we have developed formulations that reflect airway conditions in health and those that reflect the altered environment of the CF respiratory tract. Here, we describe the development and validation of these media and their use for study of genetic and phenotypic adaptations in P. aeruginosa during growth under upper or lower airway conditions in health and in CF.",
"keywords": [
"Cystic Fibrosis (CF)",
"sinuses",
"lungs",
"in vitro models",
"3Rs"
],
"content": "\n\n\n\nScientific benefit(s):\n\nAllows:\n\n• Study of bacterial pathogenicity in infection relevant conditions of compartmentalised airway niches\n\n• Study of bacterial host adaptation in cystic fibrosis (CF) and other respiratory diseases\n\n3Rs benefit(s):\n\n\n\n• Reduces the need for vertebrates in respiratory microbiology by providing a pre-screening platform for bacterial pathogenicity and host-pathogen interaction related research questions\n\nPractical benefit(s):\n\n\n\n• Inexpensive, simple to operate, no training required\n\n• Chemically defined and easy to manipulate the content of the media to study different research questions, with several respiratory pathogens\n\nCurrent applications:\n\n\n\n• Suitable for studying phenotypes including bacterial growth, antimicrobial resistance, biofilm formation,\n\n• Suitable for studying bacterial gene expression in health and CF under relevant host stresses\n\nPotential applications:\n\nOffers a platform for:\n\n• Studying long term evolution of bacteria in health and infection relevant media\n\n• Studying different pathogen combinations and how they affect virulence and resistance\n\n• Combining with cell or tissue-based models to fully capture the chemical and cellular characteristics of different airway niches in the lab environment\n\n• Assessing efficacy of drugs and therapeutics under relevant conditions\n\n\nIntroduction\n\nCystic Fibrosis (CF) is an autosomal recessive genetic disorder caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene, located on the long arm of chromosome 7.1 CFTR protein functions as an apical ion channel.1 CF has a range of severities, with airway disease being the main cause of morbidity and mortality.1 Defects in CFTR affect its ion transporting functions, leading to accumulation of sticky mucus on epithelial surfaces in affected organs, including the lungs, sinuses, pancreas, gastrointestinal tract, sweat glands and reproductive tract.1\n\nThe warm, humid and nutrient rich environment of CF airways supports growth of various bacteria. Pseudomonas aeruginosa is one of the most commonly isolated bacterial pathogens of the airways of people with CF (PwCF).2 P. aeruginosa is a Gram-negative aerobic/facultative anaerobic bacterium found in high abundance in external environments.2 P. aeruginosa colonization and infection of the airways is a significant contributor to morbidity and mortality in PwCF.3 When P. aeruginosa enters into the airways from an environmental source, it not only must adapt to the changes in nutritional and physiochemical status associated with the airway environment, but also to stresses exerted from intense antimicrobial pressure and the actions of immune responses, competition from resident microbiota and viscous mucus-related osmotic stresses.2 All of these factors influence the adaptation and evolution of P. aeruginosa within respiratory niches.2\n\nP. aeruginosa has a >6 Mb4 plastic genome that facilitates rapid adaptation to environmental stimuli.5 Environmental versatility is achieved through the action of regulatory systems, which collectively make up ~8% of the P. aeruginosa genome.6 Transition from colonization to acute infection and subsequent biofilm-associated chronic infection takes place as a result of changes in these regulatory systems that are key for controlling the expression of virulence factors in response to host environmental cues.7\n\nThe upper respiratory tract (and particularly the paranasal sinuses) has been suggested to be a protective niche for environmentally-acquired P. aeruginosa during early infection. Colonisation of this environment is thought to precede chronic lung infection.8,9 P. aeruginosa has been identified in sinusitis samples and a high level of similarity has been observed between paranasal and lung isolates.10 The presence of a low number of immune cells and reduced exposure to antibiotics in the upper airways, relative to lungs, provides a transitional niche within which the bacteria can adapt to host conditions before establishing chronic lung infection. Early infection of lungs with environmental P. aeruginosa may be cleared by a combination of host inflammatory responses and antibiotic treatment, but the persistence of the pathogen within the upper airways offers opportunity for later downward reseeding of a host-adapted bacterial population. Armbuster et al. documented the role of the sinuses in P. aeruginosa within-host adaptation in 106 adult CF patients. They found that the sinuses harboured isolates with pathoadaptive characteristics that were previously seen in isolates from sputum and that have been associated with persistence in the CF lung.11 The continuous cycle of bacterial clearance from lungs and recolonization from sinuses may form an important part of the intermittent colonization stage that precedes chronic lung infection.12\n\nThis proposed route to lung infection was recapitulated in a mouse natural inhalation infection model with P. aeruginosa, where persistence of bacteria in the upper airways was observed over the course of 28 days of infection. Initial colonisation of lungs was cleared within two weeks of infection, in the absence of antibiotic treatment, but P. aeruginosa was isolated from sinuses throughout infection and reappeared in the lungs of all mice at day 28 post-infection, suggesting that long-term upper airway persistence may allow bacterial migration and reseeding to the lower airways.8 In later studies, this within-host adaptation was linked to loss-of-function mutations in the two component regulatory system gene pmrB, thought to have been acquired during upper airway infection.13 Isolates carrying pmrB mutations, recovered from the murine airways, showed enhanced potential for establishment and maintenance of lung infection that was linked to increased resistance to lysozyme and increased attachment to host surfaces. Similar loss-of-function mutations were identified in pmrB in clinical isolates from CF. As the experiments undertaken in mice did not involve antibiotic treatment, the selective pressure for the observed changes was hypothesised to be the result of the action of host-derived antimicrobials.13\n\nOther pathoadaptive characteristics of strains from chronic infections have been identified and include overproduction of alginate, reduction in growth rate, loss of motility, decreased susceptibility to antimicrobials and diversification in colony morphology.14–16 Changes in colony morphologies, towards a so-called ‘wrinkly’ phenotype, were indicated to be the result of redox-drive adaptations to maximize oxygen accessibility in biofilm by increasing the surface area exposed to air.17 A slow growing phenotype has also been suggested to be a common characteristic of long term CF airway colonization and is thought to develop as a result of continuous activation of stress responses driven by exposure to cells and molecules of the immune system.18\n\nThe respiratory tract is a heterogenous, compartmentalised environment, with the biological, chemical and physical properties of each niche shaping P. aeruginosa adaptive evolution in different ways. A range of in vitro and in vivo models are available for the study of respiratory infection in CF. The most basic of these involve in vitro culture in nutrient rich laboratory media that readily supports bacterial growth. These models are often a poor reflection of the airway environment, as they contain sugars and proteins at concentrations that are not physiologically relevant and they lack important host-derived factors that exert stress on pathogens or that are recognised by microbial environmental sensing systems. These limitations encouraged the development of chemically-defined liquid culture media that better reflect the conditions of the CF airways. Artificial sputum media (ASM), synthetic cystic fibrosis media (SCFM) and their derivatives19–21 capture important chemical aspects of the CF airway environment, including extracellular DNA (eDNA), amino acids and mucins.20,21 These media support infection-relevant biofilm formation and have been of significant value to the research community. Furthermore, they can be combined with host tissue to provide a physical scaffold for biofilm formation.22 However, these models still lack key molecules of the lung, including host-derived antimicrobials,23,24 polyamines25,26 and a number of factors associated with CF comorbidities, including glucose, which is elevated in lungs in CF-related diabetes compared to healthy individuals,27 and bile salts, which can be inhaled into lungs during CF-associated gastro-oesophageal reflux.28\n\nMost currently available in vitro and in vivo models focus on capturing conditions of the lung environment and therefore do not reflect the heterogeneity of the airways and are unsuitable for studying early infection processes that may take place in the sinuses.21,29 Whilst in vivo infection models – most often performed in mice or rats - allow for a more holistic view of infection processes,30 they are costly, hard to maintain and there are important differences in host-derived antimicrobial structure and function between rodents and man.31 Furthermore, CF rodent models don’t reproduce the disease phenotypes and severity of human CF.30 In particular, they poorly replicate the severe pathology of CF lung disease, with the major disease phenotype instead manifesting in the gastrointestinal tract.32 The generation of gut-corrected CFTR transgenic mice has addressed this issue, but establishing chronic lung infection in these mice remains a challenge.32 Embedding bacteria within agarose beads and instilling these into the trachea of mice allows for establishment of long-term infection,33 albeit by an artificial route that requires surgical procedures and which is hard to standardise. Natural inhalation of CF-adapted P. aeruginosa suspended in saline offers an alternative that recapitulates the sinus to lung infection route and allows for long-term infection studies,8 but this model has other drawbacks, including a relatively low density of infection in lungs. Study of major pathophysiological CF characteristics, such as chronic respiratory infection, inflammation, and mucus plugging, have been hampered by the limitations of existing in vivo models.32\n\nAccording to Home Office Report from 2021, 3.06 million procedures were performed involving living animals for scientific purposes.34 More than half of these procedures were carried out in mice (54%) and 51% of overall procedures were for basic research while 27% was for applied research including human infectious disorders.34 Moreover, in the field of respiratory microbiology, 10,540 publications report the use of mice for infection (Pubmed search “respiratory infection mouse” 2015-2022), more than half of these being for bacterial infections (6,274). Development of well-characterised respiratory mimicking media would offer opportunities for both replacement and reduction in such studies.\n\nWith the aim of understanding the applicability of available in vivo and in vitro models to study human infections, Cornforth et al developed a computational framework that uses transcriptomic data from P. aeruginosa grown under different environmental conditions and benchmarks it against the gene expression patterns observed in clinical CF sputum samples. This approach has been used to assess the clinical relevance of in vitro and in vivo models, with results suggesting that in vitro models are as good as or better than mouse lung infection models at mimicking the P. aeruginosa transcriptome in human CF sputum.35\n\nGiven the limitations of the available animal models for study of CF infection and bacterial within-host adaptation, we sought to develop novel culture media that mimics the conditions of compartmentalised airway regions. To enable study of early infection processes taking place in the upper airways, we designed sinus-mimicking media reflective of conditions in health or in CF (healthy sinus media [HSM] and CF sinus media [CFSM]). In parallel, we developed media reflective of the lungs in health or CF (healthy lung media [HLM] and CF lung media [CFLM]). The developed CF media will be of use for the study of CF pathogens under infection-relevant conditions, whilst the healthy media equivalents can be used for study of airway infection in other contexts, such as non-CF bronchiectasis, community-acquired or ventilator-acquired pneumonia, that occur without the underlying altered environment that is specific to CF. Many of the differences between airway niches and between health and CF that we aim to capture have been described previously, including in O2/CO2 levels, temperature,36 lysozyme,23 lactoferrin,23 polyamines,25,26 mucins,37 amino acids, carbon source content21 and salt concentrations.38 These media were designed to be cheap, accessible and readily modifiable, according to the research question being asked. Development of validated culture media that are more relevant than using nutrient broth and more cost effective and ethical than animal models can offer a platform to understand bacterial within-host adaptation, to use for isolate virulence or antimicrobial susceptibility testing, for efficacy testing of new therapeutics, or for study of bacterial growth, gene expression and biofilm formation under relevant environmental conditions.\n\n\nMethods\n\nMicrobial strains, base media and culture conditions\n\nAll P. aeruginosa isolates used in this work are shown in Table 1. Bacteria were routinely streaked onto Tryptone Soy Agar (TSA) (Sigma-Aldrich) from bead stocks and restreaked every other day to maintain a fresh stock or from bead stocks, as required. After 18 hours of growth on agar, overnight cultures were prepared in 5ml Lysogeny Broth (LB) (Neogene) at 37°C and incubated for 18 hrs at 180 rpm. M9 medium (1 litre) was prepared by mixing 700 ml M9 salts (15 g/l KH2PO4, 64 g/l Na2HPO4, 2.5 g/l NaCl, 5.0 g/l NH4Cl) (Sigma-Aldrich) (autoclaved), 2 ml MgSO4 (autoclaved) (Sigma-Aldrich) and 20 ml 20% glycerol (Sigma-Aldrich) (filter-sterilized), in order. Sterilized water was then used to make up the volume to 1 L.\n\nSingle chemical growth rate assays\n\nEach chemical under consideration for inclusion in the respiratory tract-mimicking media was first assessed by supplementation individually into M9 media at ‘low’, ‘ideal’ or ‘high’ concentrations. Ideal concentrations represent those estimated to most closely reflect the physiological concentration found in each niche, in either health or disease. The relevance of the final list of chemicals included in the final media, in the context of bacterial adaptation to airway environments, is summarised in Table 2. The tested concentration ranges were determined by reference to the literature or from experimental measurement of metabolites (Table 3). Low and high concentration values were set at 2-fold below and above the ideal concentration of each chemical. Growth rates of PAO1 and LESB65 were assessed in M9 media supplemented with each chemical, using a microplate reader (Varioskan® LUX, Thermo Scientific) (Underlying data Figures 1-4). 2 μl of bacteria from overnight cultures were added to 198 μl of M9 media supplemented with the appropriate chemical. The bacteria were then incubated in sterile 96-well plates (U-bottom) (Greiner) for 20 hours and the growth rates assessed at OD600nm at 10 minutes intervals. Incubation conditions were 37°C/5% CO2 for lung conditions and 34°C/0% CO2 for sinus conditions. Three biological replicates were performed, per bacterial isolate, per condition. A single working concentration to be used in the final pooled media was then chosen for each chemical, per condition and growth rate assays were performed again with the chosen concentration before the chemicals were combined into a single pooled media. The ‘ideal’ concentration was chosen for the final media unless found to cause complete inhibition of bacterial growth.\n\n\n\n• Lysozyme\n\n• Lactoferrin\n\n\n\n• The effectiveness of lysozyme reduces in biofilms and in the chronically infected lung, due to electrostatic sequestration of the enzyme by infection-associated anionic biopolymers33,34\n\n• Pseudomonas aeruginosa acquire mutations leading to resistance against lysosomal killing in the lung.10\n\n• In CF, activity of lactoferrin can be inhibited by causing partial cleavage via high concentration of cathepsin (neutrophil elastase) and Pseudomonas elastase secretions.65\n\n\n\n• Spermidine\n\n• Spermine\n\n• Putrescine\n\n\n\n• P. aeruginosa utilizes host-derived polyamines to facilitate antimicrobial tolerance.25\n\n• Spermidine was shown to readily coat the surface of PmrB-deficient P. aeruginosa, protecting P.aeruginosa from antibiotics and oxidative stress and leading to increase in biofilm formation.25\n\n\n\n• The abnormal mucin glycosylation in CF promotes bacterial adhesion and infection, via increased exposure of specific glycan receptors.66\n\n• P. aeruginosa can break down and utilize mucin glycans as monosaccharides.67\n\n\n\n• Necrosis products of neutrophils and component of extracellular polymeric substance (EPS) in biofilms.\n\n• Presence of neutrophils in infection leads to increased biofilm formation as result of formation of eDNA-actin polymers.68\n\n• Bacterial eDNA can also be secreted by P. aeruginosa to aid biofilm formation.69\n\n• eDNA can shield P. aeruginosa biofilms against aminoglycosides and protect them from antimicrobial attack.69\n\n• Presence of eDNA leads to formation of cation gradients and inducible antibiotic resistance in CF airways.70\n\n\n\n• Bacteria exoproducts or derived from the host.\n\n• P. aeruginosa secretes exoproteases to generate more utilisable nutritional substrates to support its growth.71\n\n• Increased amino-acid content in the airways of CF patients plays a significant role in the selection and maintenance of nutritionally deficient P. aeruginosa.71\n\n• In the presence of amino acids, P. aeruginosa quorum sensing activity and exopolysaccharide formation increases.72\n\n\n\n• Binding of albumin can promote bacterial survival at the epithelial cell surface.53\n\n• Albumin influences formation of P. aeruginosa virulence factors. Serum albumin shown to play a critical role in P. aeruginosa virulence during early phases of infection by enhancing the expression of iron-controlled genes.73\n\n\n\n• Calcium Chloride (CaCl2)\n\n• Magnesium Chloride (MgCl2)\n\n• Iron Chloride (FeCl2)\n\n• Copper Chloride (CuCl2)\n\n• Zinc Chloride (ZnCl2)\n\n\n\n• Iron and zinc aid bacterial growth and are essential nutrients for bacteria56\n\n• Magnesium has been proposed to have a role in maintaining established biofilms56\n\n\n\n• Sialic acid\n\n• Galactose\n\n• N-acetyl glucosamine\n\n• Glucose\n\n\n\n• Products of mucin degradation in CF airways, act as nutrient sources.54\n\n• Glucose elevated in lungs in CF-associated diabetes.40\n\n\n\n• Sodium Chloride\n\n• Bile salts\n\n• Succinate\n\n\n\n• High concentrations of sodium chloride in the airway surface liquid (ASL) inhibit the activity of antimicrobial factors giving higher chance for bacterial persistence.74\n\n• Long term bile exposure shown to cause emergence of adaptive P. aeruginosa variants that are ecologically competitive sub-populations.75\n\n• Succinate-exposed P. aeruginosa showed increase in glucose metabolism and utilization of trehalose and acetate, production of EPS and enabled tolerance to oxidant stress76\n\nAmino acid concentration sources: healthy77: CF21: (x: chemical not present).\n\nMedia synonyms:\n\nHealthy sinus media (HSM),\n\nHealthy lung media (HLM),\n\nCF sinus media (CFSM),\n\nCF lung media (CFLM).\n\nMedia components were pooled together at concentrations given in Table 3, yielding four different respiratory media: Healthy sinus media (HSM), healthy lung media (HLM), CF sinus media (CFSM) and CF lung media (CFLM). Briefly, eDNA and mucin solutions were prepared separately in distilled water by constant stirring overnight at 4 °C. Next day, the solutions were autoclaved at 121 °C. Other components of the media were prepared in a single solution by firstly adding the M9 media components (water, 20% glucose and 1M MgSO4). Stock solutions of all the media components except glucose, succinate, N-acetyl glucosamine (Glc-NAc), bile, NaCl, albumin and amino acids were prepared and added to the media in solution form. Aforementioned chemicals were then added in powder form, slowly, under constant stirring at room temperature. Once all the components were fully mixed, the solution was sterilized by filtration using Vacuubrand ME 2 diaphragm vacuum pump and Millipore Steritop filter units with a pore and neck size of 0.22 μm. Sterile eDNA and mucin was then added to the filtered media and the pH of media was adjusted to 6.8-6.9 using sodium hydroxide (NaOH) (Sigma-Aldrich). The volume of the solution was brought to 1 litre by the addition of autoclaved distilled water. The media was then aliquoted to 50ml falcon tubes and stored at -80°C until further use.\n\nM9 media was prepared without the addition of M9 salts because exogenous salts were added in the form of NaCl and bile salts, as defined in Table 3. Salinity of the media was adjusted according to the salinity of ASM (2%) (measured in this study) for CF media. Salinity of healthy media was kept lower than CF media. Briefly, 1ml of media (ASM, HSM, HLM, CFSM, CFLM) was deposited to the surface of refractometer (V-RESOURCING) after it was calibrated with sterile water. Salinity of CF media was adjusted to 2%, salinity of healthy media was kept less than 2% as CF airways is known to have higher salt content than healthy airways, although salinity in health is not well defined.\n\nViscosity of media was measured before and after allowing bacteria to form biofilms for three days, to mimic chronic infection. Viscosity measurements (mPa.s) were performed using a rheometer (Anton-Paar) with cone plate (Serial number: 44806). 50-100 1/s range was used for all sterile media and healthy media during infection, and 0.01-1000 1/s range was used for CF media during infection. Changes in media viscosity was observed under each shear rate point.\n\nStability of developed media was tested over a 30-day period by performing crystal violet stain assays (as described below) using media stored at 4°C for 30 days. This experiment was designed to determine whether media would continue to support biofilm formation after prolonged storage.\n\nColony forming unit (CFU) assays were performed to observe the growth of PAO1 and LESB65 in the pooled media. Bacterial cultures grown in LB for 18 hours were centrifuged for 12 minutes at 3220rcf at room temperature. After the LB suspension was removed, bacterial pellets were resuspended in 5 ml of sterile phosphate buffer saline (PBS) (Sigma-Aldrich). The suspension was homogenized by vortexing and bacterial optical density was adjusted to OD 0.05-0.06 in PBS using an optical spectrometer (HANNA instruments). 50 ul of this suspension was then added to 4950 ul HSM, HLM, CFSM or CFLM and cultures were incubated under niche-appropriate conditions: sinus media at 34°C with no CO2, lung media at 37°C with 5% CO2. Anaerobic jars were used to increase the CO2 concentration for lung conditions. CFU was measured at pre-determined time intervals by serial-dilution onto agar. Briefly, 10-fold serial dilutions were prepared in 96 well microplates (Greiner U-bottom) using PBS as the diluent. 20 ul of bacterial dilution was then plated to TSA plates covering the dilutions from 10-2 to 10-9. The plates were air dried and then incubated at 37°C until the colonies were clearly visible (18 hours for PAO1 and 24 hours for LESB65).\n\nMeasurement of attached biofilm biomass by crystal violet staining\n\nStarting from an overnight liquid culture in LB (incubated for 18 hours), bacterial cultures were diluted 1:100 in the developed media, or in LB, or M9, two widely used laboratory bacterial culture media. To minimise edge-effect, non-perimeter wells of U-bottomed polystyrene 96-well microtiter plate (Greiner U-bottomed) were inoculated with 180 μl of these dilutions and perimeter wells were used as a negative control (sterile medium). Following 3 days of growth at 37°C, the supernatant (containing non-adhered cells) was removed from each well and plates were rinsed using 200 μl sterile PBS, twice. Subsequently, 200 μl of Crystal Violet (CV) was added to non-perimeter wells and plates were incubated at room temperature for minimum 20 minutes. After 20 minutes, the excess CV was removed by washing the plates under running tap water. Finally, bound CV was released by addition of 200 μl of 95-100% ethanol (Sigma-Aldrich) to the wells with bacteria. After incubating with ethanol for 30 minutes, the absorbance was measured at 600 nm using a BMG plate reader. 95-100% ethanol was used as a blank.\n\nMeasurement of cell viability and free-floating biofilm formation by resazurin assays\n\nOvernight cultures of PAO1 and LESB65 were diluted in LB to an OD 600 of 0.05 (±0.01). These cultures were then further diluted in the developed media (1:100) to a total volume of 10ml in glass universal tubes. Free floating biofilm formation in respiratory tract-mimicking media was compared to that observed in LB and M9. Diluted developed media cultures were incubated under conditions appropriate for each respiratory niche, as described above. Cultures were incubated for 3 days while shaking at 75 rpm. Three glass universal tubes containing bacteria-free respiratory media, LB or M9 were used as a negative control. After the incubation, biofilms were disrupted using 250 μl of 100 mg/ml cellulase (diluted in 0.05 M citrate buffer [9.6 g/l Citrate.H2O (VWR)] in water and pH to 4.6 with NaOH) and further incubated under aerobic conditions at 37 °C, while shaking at 150 rpm for 1 h. After 30 minutes incubation, biofilms were further disrupted by manual pipetting to ensure complete disruption of biofilms. A portion of disrupted biofilms were then transferred to 96-well plates (Greiner U-bottomed) and to determine the metabolic activity of the bacterial cells released from the disrupted biofilms, 10 μl of 0.02 % (v/v) resazurin (Sigma-Aldrich) (diluted in distilled water) was added to each well of the 96-well plates and incubated for 1-2 hours at niche specific temperature, while shaking at 150 rpm. Following incubation with resazurin, the fluorescence of each well was measured using an excitation wavelength of 540 nm and an emission wavelength of 590 nm in a Fluostar Omega microplate reader and the MARS Data Analysis Software.\n\nThe bacteria were grown in 5ml of each developed media or LB until early stationary phase (12 hrs for PAO1 and 20 hrs for LESB65 in the developed media, and 6hrs for PAO1 and 18hrs for LESB65 in LB). To extract RNA, TRI reagent (ZYMO Research) was added and mixtures were incubated for 5–10 min at room temperature. Bacteria were harvested by centrifugation for 30 min at 13,000 rpm and 4 °C. Supernatant was removed and bacterial pellets were used for subsequent RNA isolation or stored at −80 °C. RNA from bacterial cultures was isolated using the Direct Zol RNA Microprep kit (ZYMO Research: R2061) according to the manufacturer’s instructions. DNase digestion treatment was performed using DNAse 1 (ZYMO Research) as per the manufacturer’s protocol. Isolation was performed in an RNase-free environment using RNase-free consumables and reagents. Purified RNA was eluted with RNase-free water (ZYMO Research). Quantification of RNA was performed by measuring the absorbance at 260 nm wavelength using the NanoDrop8000 UV–vis Spectrophotometer (Thermo Scientific). and purity of RNA was analysed by determination of the 260/280 nm ratio. Nuclease free water (Invitrogen) was used as a blank. The 260/280 nm ratio obtained for all samples was between 1.8 and 2.0.\n\nFirst-strand cDNA synthesis was performed using the iScript cDNA synthesis kit (BIO-RAD: 1708891) according to the manufacturer’s instructions in an RNase-free environment using RNase-free consumables and reagents. For each sample, 2.5 ng RNA was added and incubated in a thermocycler (Applied Biosystem) under the following protocol: 5 min at 25 °C, 30 min at 42 °C and then 5 min at 85 °C. The cDNA was then stored at −20 °C until further use.\n\ncDNA was used for quantitative real-time PCR (qPCR) in duplicate reactions using the GoTaq® qPCR Master Mix (Promega: A6001) as per the manufacturer’s instructions. Each reaction contained 2 μl of cDNA (or nuclease free water for the no template control) and 0.2 μM of forward and reverse primers (Eurofins). Primer sequences are provided in Table 4. qPCR was performed using the BioRad CFX Connect Real Time PCR System (BIORAD) using MicroAmp™ Optical 96-Well Reaction Plate (Applied Biosystem) under the following conditions; 2 min at 95 °C followed by 40 cycles of 15 sec at 95 °C and 1 min at 60 °C.\n\nEach qPCR was performed with three biological replicates and in duplicate on each run. Gene rpoD, encoding sigma factor RpoD, was used as a reference gene. cDNA obtained from cultures grown in LB was used as a control. Analysis of relative gene expression in each developed media and SCFM2 versus (vs) LB was performed using the 2−ΔΔCt relative quantification method as described previously.39\n\nPAO1 was cultured in the developed media for forty days, sub-cultured to fresh media every 2 days and plated on Tryptone Congo red/Coomassie blue Agar (TCCA) prepared by mixing 20 mg/L Coomassie blue (Sigma-Aldrich), 40 mg/L Congo red (Sigma-Aldrich), 10 g/L Tryptone (Sigma-Aldrich) and 12 g/L Bacto agar (Fisher-Scientific) in distilled water and autoclaving at 121°C for 15 minutes. 100 μl of bacterial cultures were spread on to the plates in serial dilutions (10-4, 10-6) every ten days. Plates were incubated at 37°C for 24 hours and for a further 48 hours at room temperature to allow the colonies to uptake the two dyes in the media.\n\nAll assays were carried out at least three times independently. Statistical significance was evaluated using One-way ANOVA (*p<0.05, **p<0.01, ***p<0.001, **** p<0.0001). All statistical analysis was done using Graphpad Prism versions 8 and 9 (similar analysis could be performed in Microsoft Excel as a free to use alternative). For single chemical growth rate assays, R studio version 3.6.2 and GrowthCurver (version 0.3.1) was used to plot the area under the curve (AUC_e) values.\n\nHere we describe, step-by step, the procedure used to prepare 1L of each of the four developed media. Reagents and equipment used in this study are listed in Table 16.\n\nPreparation of the base (same for both CF media)\n\n1. In a 1 litre Duran bottle, add 300 ml distilled water, 20 ml 20% glucose and 2 ml 1 M MgSO4.\n\nPreparation of CF sinus media\n\n1. Add 4.8 g eDNA in 300 ml water under continuous stirring with a magnetic stirrer at room temperature. Add the eDNA very slowly as it can form clumps in the water.\n\n2. Add 6 g mucin from porcine stomach (Type II) in 150 ml water under continuous stirring with a magnetic stirrer at room temperature. Add mucin powder slowly to water to prevent the formation of clumps. eDNA and mucin can be stirred overnight at 4°C to fully dissolve the powder.\n\n3. Once the eDNA and mucin solutions are fully dissolved, autoclave at 121°C for 15 minutes to sterilise.\n\n4. Prepare stocks of lysozyme, lactoferrin, spermidine, spermine, putrescine, CaCl2, MgCl2, FeCl2, CuCl2, ZnCl2, sialic acid and galactose in the concentrations given in Table 5 for CF sinuses by dissolving each powder in deionised water.\n\n5. Under constant stirring with magnetic stirrer, from the stocks prepared, add the amounts of each chemical to the base media as given in Table 6.\n\n6. Add 35 μl of neat spermidine solution (9.296 g/l) to the base media.\n\n7. Weigh the amounts of L-amino acids given in Table 3 and add one by one to the media under constant stirring. Exceptions are L-cysteine and L-tyrosine. Dissolve L-cysteine in 2.95 ml of 0.5 M potassium hydroxide (Mr 56.11 g/mol) and L-tyrosine in 2.95 ml sterile water, separately before adding them to the base media. (Amino acid concentrations are same for both CF sinuses and CF lung media.)\n\n8. Weigh 7 g of albumin, 6.3 g of NaCl, 4.4 g of N-acetyl glucosamine, 250 mg of glucose and 2.95 g of succinate and add to the media one at a time. Wait for each chemical to dissolve completely before adding the next one. Succinate can take time to dissolve in water. Incubate the succinate at 37 °C while shaking at 180 rpm for 10-15 minutes to quicken dissolving.\n\n9. Sterilise the media by filtration using a Vacuubrand ME 2 diaphragm vacuum pump and Millipore Steritop filter units with a pore and neck size of 0.22 μm.\n\n10. Under sterile conditions, add 250 ml of autoclaved eDNA and 125 ml of autoclaved mucin to the mixture.\n\n11. Adjust pH to 6.8-6.9 with 5 M NaOH and bring the volume to 1 litre with sterile water.\n\n12. Filtered media should be stored at 4 °C for further use. Using fresh media is recommended, however it can be kept under these conditions for a maximum of three weeks. For freshness, the media can be aliquoted and stored at -80°C (suitable for longer term use).\n\nPreparation of CF lung media\n\n1. Follow steps 1-3 of CFSM protocol for eDNA and mucin stock solution preparation.\n\n2. Prepare stocks of lysozyme, lactoferrin, spermidine, spermine, putrescine, CaCl2, MgCl2, FeCl2, CuCl2, ZnCl2, sialic acid and galactose in the concentrations given in Table 7 for CF lungs by dissolving each powder in deionised water.\n\n3. Under constant stirring with magnetic stirrer, from the stocks prepared, add the amounts of each chemical to the base media as given in Table 8.\n\n4. Add 37.6 μl of neat spermidine solution (9.296 g/L) to the base media.\n\n5. Add amino acids in the same way as for preparation of CF sinuses media (See Table 3).\n\n6. Add 7 g albumin, 6.3 g NaCl, 4.4 g of N-acetyl glucosamine, 250 mg of glucose, 2.95 g of succinate, 300 mg bile salts, one at a time.\n\n7. Sterilise the media in the same way to CF sinuses media (see above protocol).\n\n8. Under sterile conditions, add 250 ml of autoclaved eDNA and 125 ml of autoclaved mucin to the mixture.\n\n9. Adjust pH to 6.8-6.9 with 5M NaOH and bring the volume to 1 litre with sterile water.\n\n10. Filtered media should be stored at 4 °C for further use. Using fresh media is recommended, however it can be kept under these conditions for a maximum of three weeks. For freshness, the media can be aliquoted and stored at -80°C (suitable for longer term use).\n\nHealthy sinuses media\n\nPreparation of the base media (Same for both healthy media)\n\n1. In a 1 litre Duran bottle, add 250 ml distilled water, 20 ml 20% glucose and 2 ml 1 M MgSO4.\n\nPreparation of healthy sinuses media\n\n1. Add 1 g eDNA in 500 ml water under continuous stirring with a magnetic stirrer at room temperature. Add the eDNA very slowly as it can form clumps in the water.\n\n2. Add 1.4 g mucin from porcine stomach (Type II) in 140 ml water under continuous stirring with a magnetic stirrer at room temperature. Add mucin powder slowly to water to prevent the formation of clumps. eDNA and mucin can be stirred overnight at 4 °C to fully dissolve the powder.\n\n3. Prepare stocks of lysozyme, lactoferrin, spermidine, spermine, putrescine, CaCl2, MgCl2, FeCl2, CuCl2, ZnCl2, sialic acid and galactose in the concentrations given in Table 9 for healthy sinuses by dissolving each powder in deionised water.\n\n4. Under constant stirring with magnetic stirrer, from the stocks prepared, add the amounts of each chemical to the base media as given in Table 10.\n\n5. Add 21.5 μl of neat spermidine solution (9.296 g/l) to the base media.\n\n6. Add amino acids in the same way as for preparation of the CF media, see Table 3.\n\n7. Add 500 mg albumin, 1g NaCl, 1.28 g of N-acetyl glucosamine, one at a time.\n\n8. Sterilise the media in the same way as for CF sinuses media (see above protocol)\n\n9. Under sterile conditions, add 480 ml of autoclaved eDNA and 120 ml of autoclaved mucin to the mixture.\n\n10. Adjust pH to 6.8-6.9 with 5 M NaOH and bring the volume to 1 litre with sterile water.\n\n11. Filtered media should be stored at 4 °C for further use. Using fresh media is recommended, however it can be kept under these conditions for a maximum of three weeks. For freshness, the media can be aliquoted and stored at -80 °C (suitable for longer term use).\n\nHealthy lungs media\n\nPreparation of healthy lungs media\n\n1. Follow steps 1-3 of HSM protocol for eDNA and mucin stock solution preparation.\n\n2. Prepare stocks of lysozyme, lactoferrin, spermidine, spermine, putrescine, CaCl2, MgCl2, FeCl2, CuCl2, ZnCl2, sialic acid and galactose in the concentrations given in Table 11 for healthy lungs by dissolving each powder in deionised water.\n\n3. Under constant stirring with magnetic stirrer, from the stocks prepared, add the amounts of each chemical to the base media as given in Table 12.\n\n4. Add 26.8 μl of neat spermidine solution (9.296 g/l) to the base media.\n\n5. Add amino acids in the same way of preparation of the CF media, see Table 3 for concentrations.\n\n6. Add 1.5 g albumin, 1 g NaCl, 1.28 g of N-acetyl glucosamine, one at a time.\n\n7. Sterilise the media in the same way to CF sinuses media (see protocol for CF sinuses)\n\n8. Under sterile conditions, add 480 ml of autoclaved eDNA and 120 ml of autoclaved mucin to the mixture.\n\n9. Adjust pH to 6.8-6.9 with 5 M NaOH and bring the volume to 1 litre with sterile water.\n\n10. Filtered media should be stored at 4 °C for further use. Using fresh media is recommended, however it can be kept under these conditions for a maximum of three weeks. For freshness, the media can be aliquoted and stored at -80 °C (suitable for longer term use).\n\nIncubation conditions for the media\n\nIncubation conditions differ between lung- and sinus media. The incubation conditions for each media are given in Table 13. Conditions of 5% CO2 can be achieved by incubating the media in GasPak Jars with candles, using Campygen packs or microaerobic incubators\n\nNotes on handling of media components and media preparation\n\n• Break bovine serum albumin into smaller pieces before adding to media, as it can take time to fully dissolve.\n\n• Add eDNA and mucin to minimise clumping. Make sure that each addition is dissolved before adding more.\n\n• Succinic acid may not fully dissolve in water immediately. Leave it at 37 °C for 30 minutes if needed.\n\n• Polyamines should be handled in a fume hood due to toxicity at high concentrations. Read supplier instructions carefully.\n\n• If a sterile pH probe cannot be used, we recommend aliquoting a small volume of media (2 ml) before each measurement, to maintain media sterility. Discard the aliquoted media after taking a measurement.\n\n• As CF media is rich in chemicals, it is advised to use more than one filter unit, to prevent saturation.\n\n\nResults\n\nTesting the effect of individual chemicals on bacterial growth\n\nFor each chemical under consideration for inclusion in respiratory-mimicking media, the effect on P. aeruginosa growth was determined at three different concentrations. Experiments were performed over 20 hours of growth for PAO1 and LESB65, using M9 media as a base to support minimal bacterial growth (Underlying data Figures 1-4). PAO1 was chosen as a non-CF laboratory reference strain, to act as a comparator to the CF-adapted strain LESB65. Chosen concentrations of all chemicals were higher in CF conditions vs health conditions. Most chemicals were included in both health and CF conditions, with the exception of bile salts and glucose, which were only tested in CF conditions. Airway glucose is significantly elevated in CF related diabetes and bile in the lower airways is associated with CF, as a co-morbidity of gastroesophageal reflux disease.28,40\n\nResults from these experiments showed that, when used individually, most of the tested chemicals enhanced growth of both PAO1 and LESB65 (Underlying data Figures 1-4). The highest concentrations of bile salts, succinate and extracellular DNA (eDNA) used in CF conditions were toxic to bacteria (Underlying data Figures 1-4 (CF lung and CF sinus graphs)). The antimicrobial activity of lysozyme and lactoferrin was also observed to be both niche and concentration dependent, potentially due to decreased antimicrobial activity in lower temperatures, charge interference at high concentrations or induction of bacterial defence mechanism.\n\nPAO1 and LESB65 can be maintained in respiratory-mimicking media for at least three days\n\nNext, the chosen concentration of each chemical was used to prepare pooled media. Growth of PAO1 and LESB65 was determined in the respiratory media, by CFU determination over 72 hours (Figure 1). Data from these experiments show that viable PAO1 and LESB65 populations were maintained in all media for at least three days, with mid exponential growth of bacteria occurring at ~1×108 CFU/ml (Figure 1), similar to the findings of Palmer et al. for growth of P. aeruginosa in CF sputum.21 PAO1 was able to grow to a higher density in CF sinus and lung media than in either of the equivalent conditions in health. In addition, PAO1 grew faster than LESB65 in all media and reached early stationary phase after 12-14 hours of growth, whilst LESB65 reached stationary phase only after 24 hours of growth in each media. Although LESB65 reached a similar density in all four media, CFU began to decrease in healthy sinus and lung media after 34 hours.\n\nMedia were generated using M9 supplemented with the ideal concentrations of all the chemicals shown in tables in media preparation protocol section. These concentrations differed between sinuses and lung and between health and CF, generating four different media: Healthy sinus media (HSM), CF sinus media (CFSM), healthy lung media (HLM) and CF lung media (CFLM). CFU counts are made by taking a sample from bacterial culture grown in each developed media, every 2 hours, for 72 hours. Data shows mean of 3 biological replicates per time point, error bars show standard deviation.\n\nBoth attached and free-floating biofilm structures are thought to contribute to success of P. aeruginosa in the CF airways. Mature biofilms are commonly observed as free floating structures in mucus, whereas surface attached biofilms are suggested to dominate the early stages of infection, as physical contact of bacteria and epithelium is necessary as an initial survival strategy.41,42 Crystal violet (CV) staining assays showed that there are niche, disease state and strain specific influences on biofilm formation (Figure 2). PAO1 formed attached biofilms more readily than LESB65 in all airway niches and conditions. This may be due to the differences in growth rate of PAO1 and LESB65. In particular, PAO1 was able to form significantly more attached biofilm in sinus media compared to lung media. No statistically significant differences in biofilm formation were observed with LESB65 between healthy airway niches, but biofilm formed more readily in CF sinus media than in CF lung media. These results further highlight the importance of studying different airway niches and conditions in isolation. The media were compared with two widely-used laboratory media (LB and M9), and attached biofilm formation was shown to form most readily in LB.\n\nBacteria were left to form biofilms for 72 hours. Unattached bacteria were removed and washed with PBS to ensure removal of all unattached bacteria. Attached bacteria stained with 0.25% CV and absorbances were measured by using 95-100% ethanol as blank control at OD600. Statistical analysis is made using Graph pad Prism 8.0. Statistical analysis: One-way ANOVA *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. Each bar graph represents 3 biological replicates (mean), each with 30 technical replicates, n:90, error bars show standard deviation).\n\nFree-floating biofilm formation, associated with chronic infection, was assessed in respiratory-mimicking media, LB and M9, Figure 3. Free-floating biofilms formed more readily in LB and M9 than in any of the four respiratory media, for both isolates, suggesting that the respiratory media alter bacterial growth and biofilm formation, compared to standard laboratory media. Biofilms grew well in CF sinus and lung media, and in healthy lung media, but only minimal biofilm was observed in healthy sinus media. PAO1 outperformed LESB65 in the attached biofilm formation assays but not in the free-floating biofilm formation assays. As an airway adapted strain from chronic infection, LESB65 may be more adept at forming free-floating biofilms.\n\nBacteria were left to form biofilms for 72 hours and biofilms were disrupted with cellulase. Cultures were incubated with resazurin dye for 2 hrs. Released fluorescence was measured at 540 nm excitation λ and 590 nm emission λ. Background fluorescence of each media is corrected by subtraction of the background fluorescence obtained from the wells with media only. Statistical analysis is made using Graph pad Prism 8.0. Statistical analysis: One-way ANOVA *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. Each bar graph represents 3 biological replicates (mean), each with 30 technical replicates, n:90, error bars show standard deviation).\n\nViscosity measurements were taken in the four developed media, pre and post biofilm formation (Figure 4). Sterile HSM and HLM media were shown to have Newtonian liquid characteristics with very similar viscosity measurements to water (~1.0 mPa·s viscosity for water, 1.02 mPa·s for HSM and 1.03 mPa·s for HLM) (Figure 4A). Viscosity changed only minimally in healthy media after bacterial growth (1.06 mPa·s for HSM and 1.05 mPa·s for HLM) (Figure 4A). Both CF media were also determined to be Newtonian fluids pre-biofilm formation but had higher viscosity measurements compared to healthy media (1.37 mPa·s for CFSM and 1.27 mPa·s for CFLM) (Figure 4A). When the biofilm was established in CF media, both media became more viscous, as compared to both their sterile controls and compared to post-biofilm formation healthy media. Infected CF media showed clear non-Newtonian fluid characteristics, with shear thinning properties (viscosity decreases as the shear rate increases) (Figure 4B). Viscosity of CFSM media was approximately 16 mPa·s during infection at low shear rate ranges and decreased down to 1.57 mPa·s at the highest shear rate (1000 1/s). CFLM media was the most viscous media during infection, with approximately 2400 mPa·s viscosity at low shear rates, decreasing down to 1.8 mPa·s at 1000 1/s shear rate. These results are in agreement with previous literature, where the viscosity of CF sputum was shown to be higher than healthy controls.43\n\nViscosity of all media before infection and healthy media during infection (A) was measured under shear rate range of 50-100 1/s whereas viscosity of CFSM and CFLM media was measured at 0.01-1000 1/s shear rate range as the viscosity properties of CF media during infection was higher (B). Averages were taken for media with Newtonian fluid characteristics (All media before infection and HSM and HLM media during infection). Data points with open triangle in CFSM during infection displayed high level of uncertainty due to non-Newtonian responses and therefore values below 0.1 (1/s) should be treated with caution. Graphs are showing single viscosity measurement per media under each condition.\n\nBiofilm formation of PAO1 was measured over a month in the developed media stored at 4°C to test the stability of the airway mimicking media. Over a 30-day period, there was no significant change in the amount of biofilm formed by PAO1 in any of the four airway mimicking media (Figure 5).\n\nEach timepoint is one biological replicate representing the average of three technical replicates. No statistical significance was found between any of the fresh media vs the later treatments (One-way ANOVA *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001).\n\nFour P. aeruginosa genes were selected, to determine whether growth in respiratory-mimicking media induced CF infection-relevant gene expression. The major virulence regulator, extracytoplasmic sigma factor algU, and mexB (part of a tripartite multidrug efflux system) genes were selected as they are virulence genes of P. aeruginosa known to contribute to chronic infection.44,45 PA2911 and PA2382 genes were also selected, as expression of these genes in clinical isolates in CF sputum is observed but expression has not previously been captured in in vitro CF models.46 algU is responsible for alginate overproduction, causing mucoidy and contributing to chronic infection in PwCF.44 MexB is responsible for exporting biological metabolites and antimicrobial compounds, playing a crucial role in antimicrobial resistance in the airways.45 PA2911 is predicted to be a Ton-B dependent receptor located in the outer membrane and responsible for sideophore transport.47 PA2382 (lldA) is a L-lactate dehydrogenase, important for pyruvate metabolism.48 Results from qPCR studies showed that expression of all four genes were increased in all the airway-mimicking media when compared to expression in LB. Expression of the four genes was also determined in P. aeruginosa (PAO1 and LESB65) grown in SCFM2. Expression of algU and mexB was comparable in airway media and SCFM2, but expression of PA2911 and PA2382 was higher in respiratory-mimicking media than in SCFM2, although did not reach statistical significance, Figure 6.\n\nEach biological replicate includes the mean of two technical replicates. RNA from bacteria grown in each media was extracted and used to synthesise cDNA. qPCR was performed using the cDNA by primers specific to A and E: algU, B and F: mexB, C and G: PA2911 and D: PA2382 (for which no expression was observed in LESB65 under any conditions). cDNA from LB was used as a negative (no treatment control). Statistical analysis: one-way ANOVA.\n\nPAO1 growing in respiratory-mimicking media show changes in colony morphology\n\nPAO1 was grown for a period of 40 days in each of the four media. Every ten days, PAO1 populations cultured under different media conditions were streaked onto TCCA plates to observe colony morphotypes (Figure 7). Only shiny, circular, concave colonies were observed in the starting populations. Wrinkly colonies began to appear in all media conditions over time. These wrinkly structures have been suggested to be the result of redox-driven adaptation that maximizes oxygen accessibility in biofilms, to increase their surface area in oxidant limitation.17 Colony morphotypes in Figure 7D and E were visible only after transfer 10 and were unique to CF media (Table 14).\n\nCultures were streaked onto TCCA plates that contained Congo red and Coomassie blue to check for changes in colony morphologies. The plates were incubated at 37°C overnight and at room temperature for 48 hours before analysis. (B and C: wrinkly morphotypes, A: starting culture, D and E: colonies only observed in CF media.\n\n\nDiscussion\n\nHere we describe a simple suite of liquid culture models, designed to reproduce the conditions of upper and lower airway niches in CF and health. Our media have been designed to mimic the key properties of CF and healthy airways and have been validated for studying growth, as well as attached and free-floating biofilm formation of laboratory and CF-associated strains of P. aeruginosa. We previously showed that bacteria evolved within upper airways can acquire adaptive mutations conferring resistance to host-derived antimicrobials found in abundance in the lower airways.13 Therefore, in this work, by inclusion of different host-derived antimicrobials in the media, we aimed to capture an important source of selective pressures acting on bacteria during airway infection. We show that bacteria can be maintained and cultured in these media long term, making them suitable for studying bacterial adaptation to airway environments using experimental evolution approaches in the laboratory. We envisage these media can help reduce the need for use of animals for the purposes of experimental evolution, investigation of host-pathogen interactions and for virulence screening.\n\nTo date, different liquid culture media have been developed with the aim of capturing the conditions of CF sputum. However, these media reflect the properties of lower airway regions. The growing evidence for the importance of the upper airways in shaping bacterial within-host adaptation makes liquid culture media reflecting sinus conditions a useful addition to the CF microbiologist’s toolkit. Having bespoke media for individual airway compartments allows airway niches to be studied in isolation, something impossible to do in vivo. To our knowledge, there are also no liquid culture media available that have been developed to mimic the conditions of healthy airways. In addition to the CF media, healthy sinus (HSM) and healthy lung (HLM) media will allow the research community to study other aspects of respiratory microbiology, such as bacterial sinusitis, or pneumonia.\n\nIn respiratory microbiology a common experimental approach is to screen panels of bacterial isolates in mouse models to determine virulence.49–51 In one example study, researchers competed different pneumococcal serotypes in a mouse model of pneumococcal nasopharyngeal carriage.52 The study used >200 mice, but many experiments could have been performed in sinus mimicking media, had it been available, with only the neutrophil-depletion studies necessitating animal usage. Thus, sinus mimicking media could have enabled 60-75% reduction in usage in the study. Carriage protocols are mild-severity, but similar studies to compare virulence in moderate or severe pneumonia models49 could utilise lung mimicking media.\n\nThe culture media could be used to examine how bacterial communities develop in the face of host pressures, enabling study of biofilm formation in relevant environments. They offer a platform for assessment of bacterial gene expression in response to stress and to assess efficacy of drugs and therapeutics in a system more relevant than pathogen growth in broth, but more cost effective, less labour-intensive and ethical than animal models. The media can also be used as a pre-screening tool, to identify the most phenotypically interesting isolates to take forward into studies in more complex in vitro or in vivo systems, potentially leading to a significant reduction in the number of animals used to study bacterial pathogenicity in the host environment.\n\nThe developed media were designed to be cost effective for the purposes of long-term experiments (Table 15). The media should also prove readily accessible, as they can be prepared with standard laboratory equipment and without the need for any specialist training. The media can be easily modified to answer different research questions, for example by addition or removal of a metabolite or antimicrobial of interest. The media were also found to remain stable for 4 weeks at 4°C, although we recommend storing in -80°C for continued long term use.\n\nAt concentrations associated with conditions of the healthy sinuses (Underlying data Figures 1 and 2), PAO1 showed increased growth in the presence of only two of the chemicals (eDNA and FeCl2), relative to the M9-only control. By contrast, at concentrations associated with CF sinuses (Underlying data Figures 1 and 2), significant increases in growth were observed for six of the chemicals (lactoferrin, mucin, eDNA, albumin, amino acids and FeCl2), relative to the M9-only control. Chemicals that induced higher growth rates at one or more of the concentrations tested for healthy and CF sinuses were albumin, eDNA, amino acids, iron, magnesium, copper, zinc and N-acetyl glucosamine (GlcNac). Many of these factors are well known to promote bacterial survival and may act as nutrient sources in these niches.21,53–55 Iron and zinc are essential metals for bacteria.56 Bacteria must actively acquire them through high affinity transport mechanisms.56 In addition to these potential nutrient sources, the host derived antimicrobials lysozyme and lactoferrin were able to partially inhibit the growth of PAO1 at concentrations associated with health, however they did not cause significant growth inhibition at the higher concentrations of CF sinuses.\n\nSimilar to PAO1, increased growth of LESB65 was observed at CF-relevant concentrations for the majority of the chemicals. Growth was higher than the M9 control for at least one tested concentration of albumin, amino acids, mucin, spermine, zinc, lysozyme and lactoferrin. Relative to PAO1, the airway adapted isolate LESB65 appeared better able to grow in the face of high concentrations of host-derived antimicrobials and airway-abundant chemicals such as polyamines.\n\nOne of the key differences between CFSM and CFLM conditions was that the host-derived antimicrobial lactoferrin decreased the growth of PAO1 in CFLM while it increased the growth at all tested concentration in CFSM, where it may be acting as a nutrient source, as it is heavily glycosylated.57 The enzymatic activity of lactoferrin will not function optimally at 34°C in sinuses media (SLM), nullifying its growth inhibiting activity.\n\nLysozyme had no significant positive or negative effect on growth of P. aeruginosa in CFLM but, in CFSM, all concentrations stimulated higher rates of growth. This may be a result of lysozyme-dependent changes in bacterial phenotype. Previously, it has also been suggested that the effectiveness of lysozyme is reduced in chronically infected airways due to electrostatic sequestration of the enzyme by anionic biopolymers.58,59 Over production of inhibitor of vertebrate lysozyme (ivy) protein may also explain success in lysozyme-rich environments, as ivy knockout mutants show growth defects in the presence of lysozyme.60 eDNA elevated bacterial growth in CFLM while it showed a growth inhibiting effect in HLM, where the concentrations tested were lower, further highlighting the positive effect of the chemically rich CF airways on bacterial survival.\n\nGrowth curves of PAO1 and LESB65 in airway media suggest a doubling time for PAO1 in CF media of 74 minutes (in both CFSM and CFLM), similar to the findings of Yang et al. for PAO1 in CF sputum (66 minutes).18 The doubling time of PAO1 was ~85 minutes in healthy sinus and lung media. Doubling time of LESB65 was ~105 min in CFSM and ~92 minutes in CFL and ~86 minutes in both healthy media. This finding can be supported by the findings of Yang et al. who found two to threefold slower generation times in isolates from PwCF patients, compared to a laboratory strain. This observed reduced growth was also seen in other CF isolates, compared to non-CF isolates.18 One possible explanation for this could be that the slow-growing phenotype develops as result of the stressful conditions associated with continuous exposure to host-derived antimicrobials and other molecules of the immune system. PwCF also undergo intensive antibiotic treatment, which acts as a further stress signal. Chromosomal mutations associated with antibiotic tolerance often also have growth rate-reducing effects.61\n\nResults from phenotypic assays show that airway media induce responses in P. aeruginosa that differ considerably from common laboratory and currently available liquid culture media (such as SCFM2), as evidenced by differences in attached and free-floating biofilm formation and expression of infection-relevant genes, including those that are highly expressed in CF sputum but poorly expressed in available liquid culture models. SCFM was developed to mimic the nutritional components of CF sputum and contains amino acids, ions, glucose and lactate21 whereas HSM, HLM, CFSM and CFLM have been developed with the aim of not only capturing nutritional components but also the host derived antimicrobial content of different airway niches as well as stimulators of bacterial signalling systems, such as polyamines and bile salts.\n\nAn obvious limitation of these in vitro mimics is that they fail to capture the host cellular characteristics of different airway niches. The presence of immune cells significantly affects the pathophysiology of CF as a result of both the inflammatory processes and the direct effects of immune cells on bacteria.62 Experiments combining the developed media with cell lines or primary cells (e.g., macrophages, epithelial cells) could provide additional information about host-pathogen interactions within the airways.\n\n\nConclusion\n\nIn summary, we present here a collection of culture media replicating different airway niches in health and CF. Importantly, our study showed that these media can be used to study different phenotypic characteristics of bacteria or study of host-pathogen interactions. We show that the media can be used to study P. aeruginosa, a well-known CF pathogen, in health and disease by recapitulating different areas of the respiratory tract. With these compartmentalised airway-mimicking media, we aim to answer questions relating to how each airway niche affects bacterial behaviour and adaptation in CF and non-CF conditions, without the need for invasive whole animal studies for preliminary studies. We hope these media will become a resource for clinical microbiologists, respiratory clinicians, evolutionary biologists, pharmacologists and immunologists, as a tool to understand bacterial physiology, evolution, virulence and resistance to novel therapeutics, and as a means of reducing or replacing animal use.\n\n\nData availability\n\nFigshare: Underlying data for “Development of liquid culture media mimicking the conditions of sinuses and lungs in cystic fibrosis and health”, https://doi.org/10.6084/m9.figshare.20463159.v6.81\n\nThis project contains the following underlying data:\n\n- Biofilm assay OD values and fluorescent intensity.xlsx\n\n- F1000_qPCR_RawData.xlsx\n\n- Growth CFUs.xlsx\n\n- viscosity supplementary.xlsx\n\n- Colony E fig 7.png\n\n- colony D fig 7.png\n\n- colony C fig 7.png\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).",
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PubMed Abstract | Publisher Full Text\n\nWilton M, Charron-Mazenod L, Moore R, et al.: Extracellular DNA Acidifies Biofilms and Induces Aminoglycoside Resistance in Pseudomonas aeruginosa.2015 [cited 2020 Jun 3].Reference Source\n\nMulcahy H, Charron-Mazenod L, Lewenza S: Extracellular DNA chelates cations and induces antibiotic resistance in Pseudomonas aeruginosa biofilms. PLoS Pathog. 2008 Nov; 4(11): e1000213. PubMed Abstract | Publisher Full Text\n\nBarth AL, Pitt TL: The high amino-acid content of sputum from cystic fibrosis patients promotes growth of auxotrophic Pseudomonas aeruginosa. J. Med. Microbiol. 1996 [cited 2022 Mar 8]; 45(2): 110–119. PubMed Abstract | Publisher Full Text\n\nDiraviam D: Microbiology Insights Amino Acids enhance Adaptive Behaviour of Pseudomonas Aeruginosa in the cystic Fibrosis Lung environment.2010 [cited 2020 Jun 2].Reference Source\n\nKruczek C, Wachtel M, Alabady MS, et al.: Serum albumin alters the expression of ironcontrolled genes in Pseudomonas aeruginosa. Microbiology. 2012 Feb 1 [cited 2022 Mar 8]; 158(2): 353–367. PubMed Abstract | Publisher Full Text\n\nKhubchandani KR, Oberley RE, Snyder JM: Effects of Surfactant Protein A and NaCl Concentration on the Uptake of Pseudomonas aeruginosa by THP-1 Cells.2012 Dec 14 [cited 2022 Mar 8]; 25(6): 699–706. Publisher Full Text Reference Source\n\nFlynn S, Reen FJ, O’gara F: Exposure to bile leads to the emergence of adaptive signaling variants in the opportunistic pathogen pseudomonas aeruginosa. Front. Microbiol. 2019; 10(AUG): 2013. PubMed Abstract | Publisher Full Text\n\nRiquelme SA, Lozano C, Moustafa AM, et al.: CFTR-PTEN-dependent mitochondrial metabolic dysfunction promotes Pseudomonas aeruginosa airway infection. Sci. Transl. Med. 2019 Jul 3; 11(499). PubMed Abstract | Publisher Full Text\n\nSchwab U, Abdullah LH, Perlmutt OS, et al.: Localization of Burkholderia cepacia complex bacteria in cystic fibrosis lungs and interactions with Pseudomonas aeruginosa in hypoxic mucus. Infect. Immun. 2014; 82(11): 4729–4745. PubMed Abstract | Publisher Full Text\n\nHenke MO, John G, Germann M, et al.: MUC5AC and MUC5B mucins increase in cystic fibrosis airway secretions during pulmonary exacerbation. Am. J. Respir. Crit. Care Med. 2007 Apr 14; 175(8): 816–821. PubMed Abstract | Publisher Full Text\n\nBlanchette KA, Shenoy AT, Milner J, et al.: Neuraminidase A-exposed galactose promotes Streptococcus pneumoniae biofilm formation during colonization. Infect. Immun. 2016; 84(10): 2922–2932. PubMed Abstract | Publisher Full Text\n\nReen FJ, Woods DF, Mooij MJ, et al.: Respiratory Pathogens Adopt a Chronic Lifestyle in Response to Bile. Kaufmann GF, editor. PLoS One. 2012 Sep 26 [cited 2020 Apr 2]; 7(9): e45978. PubMed Abstract | Publisher Full Text\n\nRuhluel D, O’Brien S, Fothergill JL, et al.: Underlying data for “Development of liquid culture media mimicking the conditions of sinuses and lungs in cystic fibrosis and health”. figshare. [Dataset].2022. Publisher Full Text"
}
|
[
{
"id": "151644",
"date": "25 Oct 2022",
"name": "Ville-Petri Friman",
"expertise": [
"Reviewer Expertise Microbiology"
],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis is a very nice manuscript describing four new synthetic media mimicking growth conditions in the human lung. The main advancement is the development of both CF and healthy lung media and the addition of host-derived antimicrobials and stimulators (bile salts and polyamines) of bacterial signalling systems that are not included in commonly used SCFM2 medium. To some extent, this comparison falls a bit short as not all measurements are performed in SCFM2 and developed media. Moreover, the gene expression analysis relies on the expression of a few marker genes instead of whole genome transcriptomics, which could have revealed further interesting differences and allowed linking data with available transcriptomics data from CF lungs. Nevertheless, these are small omissions and could perhaps just be discussed briefly in the discussion.\nMy main concern is the statistical analysis that could be improved a bit. I would suggest using full factorial analysis to clearly show if growth in the media is different for the two studied strains and if there are any interesting interactions between strain identity and specific media. These statistics should be properly reported in the text or supplementary tables including test statistics, error, and degrees of freedom.\nI would suggest:\nUse Repeated Measures Anova or GLMMs for analysing time-dependent data in Figure 1.\n\nInclude strain identity in the model in Figures 2-3 to statistically compare if strains grew differently in these media.\n\nFigure 4: Which bacteria were grown in these media before the measurements? Or is this data averaged over both bacteria? Needs a little bit more information.\n\nFigure 5: Could also analyse the effect of time for each media and across all media to show that no change in biofilm formation was observed. I would reconsider naming this measure as ‘Stability of biofilm formation’ as this is what you measure instead of media stability, which could be understood as a measure of degradation of its components. Just to avoid confusion.\n\nFigure 6: I think the results here are much more nuanced than described in the results text. For example, the expression of algU is clearly different from other marker genes and there is no clear increase in its expression relative to growth in LB media (in contrast to what is said in the results text). Here, factorial Anova (or GLMM) would be powerful to show significant interactions between different media, studied strains, and developed media.\n\nFigure 7 and Table 14: Do you have quantitative data for colony morphotype frequencies during the experiment? Data is now quite descriptive.\n\nOut of interest, did you specifically test the effect of freezing (-80°C) for media composition?\n\nAre a suitable application and appropriate end-users identified? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate? Partly\n\nAre the 3Rs implications of the work described accurately? Yes\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": "9030",
"date": "30 Nov 2022",
"name": "DILEM RUHLUEL",
"role": "Author Response",
"response": "Thank you for the helpful comments and suggestions for alternative data analysis. We have addressed each point raised, below and have submitted a revised manuscript in which the indicated changes have been made. To some extent, [the phenotypic] comparison falls a bit short as not all measurements are performed in SCFM2 and developed media. Moreover, the gene expression analysis relies on the expression of a few marker genes instead of whole genome transcriptomics, which could have revealed further interesting differences and allowed linking data with available transcriptomics data from CF lungs. Nevertheless, these are small omissions and could perhaps just be discussed briefly in the discussion. We agree that transcriptomic data would represent an important validation dataset for these media. We have now added some discussion of this point to the manuscript and hope to be able to report these data shortly. We agree, also, that it would be preferable to have SCFM2 data for all phenotypic comparisons. Where these are absent, we have now referenced studies conducted by others, in which SCFM2 was used. My main concern is the statistical analysis that could be improved a bit. I would suggest using full factorial analysis to clearly show if growth in the media is different for the two studied strains and if there are any interesting interactions between strain identity and specific media. These statistics should be properly reported in the text or supplementary tables including test statistics, error, and degrees of freedom. I would suggest: Use Repeated Measures Anova or GLMMs for analysing time-dependent data in Figure 1. We were cautious of over-interpreting these data, particularly when comparing between strains, as PAO1 and LESB65 have considerable differences in growth profile, even in standard laboratory media. However, we agree with the reviewer that comparisons of relative growth between media would be informative. We have now included empirical area under the curve (AUC_e) values for both strains, in all four media. These were determined from the original growth data in Figure 1. Two-way ANOVA analysis (alpha 0.05) of the AUC_e values shows that both strain and media type are significant variables (p < 0.0001, with 3 df for media type, p = 0.0084, with 1 df for strain). Sidak’s multiple comparison testing demonstrates the success of PAO1 in the CF media, relative to in healthy sinus and lung media. P values and a description of the findings have been added to the manuscript text and the AUC_e values are presented in Figure 1C. Include strain identity in the model in Figures 2-3 to statistically compare if strains grew differently in these media. We have done as suggested and provide updated figures, analysis and text in the revised manuscript. Strain was found to be a significant variable in both biofilm assays. We have reported summary statistics and pairwise comparisons in the text. Briefly, for crystal violet assays, PAO1 formed attached biofilms more readily than LESB65 in all airway niche media bar HLM (p < 0.0001 vs LESB65 in HSM, CFSM and CFLM, p > 0.9999 in HLM in two-way ANOVA with Sidak’s multiple comparisons test). In resazurin assays, PAO1 biofilm formation differed significantly from that of LESB65 in HLM (p = 0.0001) and HSM (p < 0.0001) but not in the other media tested. Figure 4: Which bacteria were grown in these media before the measurements? Or is this data averaged over both bacteria? Needs a little bit more information. This was done with PAO1. We have added this info to the figure legend and the text in the results section. Figure 5: Could also analyse the effect of time for each media and across all media to show that no change in biofilm formation was observed. We have re-analysed with an ordinary two-way ANOVA. Media is a significant factor (P<0.0001), whilst media storage time is not (P=0.4174). No individual pairwise comparisons between timepoints proved significant for any of the four media. We have reported the ANOVA summary statistics in the text, as suggested. I would reconsider naming this measure as ‘Stability of biofilm formation’ as this is what you measure instead of media stability, which could be understood as a measure of degradation of its components. Just to avoid confusion. The title has been changed, as suggested. Figure 6: I think the results here are much more nuanced than described in the results text. For example, the expression of algU is clearly different from other marker genes and there is no clear increase in its expression relative to growth in LB media (in contrast to what is said in the results text). Here, factorial Anova (or GLMM) would be powerful to show significant interactions between different media, studied strains, and developed media. Apologies, our interpretation of these data was overly simplistic. We have now performed two-way ANOVA for each gene, to assess the contribution of strain and media type to observed differences. We have reported these in the manuscript, along with updated graphs and data descriptions. Briefly, strain was found to be a significant variable for mexB and PA2382 expression (p < 0.0001 for both), whilst media type was a significant variable for mexB (p = 0.0308) and PA2911 (p = 0.0462) expression. In Dunnett’s multiple comparison test, with LB set as the control group for each strain, significant differences in gene expression were identified for mexB with LESB65 grown in HSM (p = 0.0249) or CFSM (p = 0.0201) and for PA2382 with PAO1 grown in CFSM (p = 0.0243) or CFLM (p = 0.0273). We agree that algU is clearly different from the other genes and have acknowledged this in the manuscript text. Figure 7 and Table 14: Do you have quantitative data for colony morphotype frequencies during the experiment? Data is now quite descriptive. Unfortunately not. We plated a proportion of the total growth at each transfer and then recorded the different colony morphotypes as present or absent, rather than a quantitative measure. Out of interest, did you specifically test the effect of freezing (-80°C) for media composition? Only in the same way as reported for the 4°C storage, i.e. we confirmed that the media would still support biofilm growth. We’ve added to the discussion that LCMS could provide additional useful data on media stability under different conditions."
}
]
},
{
"id": "152082",
"date": "25 Oct 2022",
"name": "Sheyda Azimi",
"expertise": [
"Reviewer Expertise Chronic infections",
"Biogeography of Infection",
"Pseudomonas aeruginosa population dynamics."
],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nIn this article, the authors sought to develop four new culturing media—Healthy Sinus Media (HSM), Healthy Lung Media (HLM), Cystic Fibrosis Sinus Media (CFSM), and Cystic Fibrosis Lung Media (CFLM)—to recapitulate the environment of human lungs and sinuses, both in healthy individuals and those with cystic fibrosis (CF). The authors justify the need for the development of novel media by emphasizing that Artificial Sputum Media (ASM) and Synthetic Cystic Fibrosis Media (SCFM and SCFM2) capture many key nutritional aspects of the CF lung, they lack critical host-derived antimicrobials, polyamines, glucose, and bile salts. All of which impact Pseudomonas aeruginosa growth. Furthermore, there are yet no media that have been developed to capture the environments of healthy human lungs or sinuses, nor that of CF-impacted sinuses.\nThis article is scientifically sound in developing and thoroughly testing four novel media to model human airways. The authors employ many different experimental tests to assay the viability of these novel media and assess their relevance to P. aeruginosa infection, including but not limited to: culturing strains in each chemical component independently before pooling, measuring growth and biofilm phenotypes, assessing long-term media stability, measuring gene expression, etc.\nThe authors use appropriate statistical tests as necessary and conduct all experiments with technical and biological replicates. In addition, the authors describe detailed methods for preparing each medium, including supplier information for all reagents, stock concentrations, volumes, final concentrations, sterilization procedures, and special notes on media preparation so that others may easily reproduce these procedures.\nThe authors provide a rationale for each component added with references to literature. The authors conduct all experiments in appropriate conditions to mimic in vivo, for example, altering growth temperatures between sinus and lung media. The article also provides sufficient rationale for the necessity of these media in the scientific community. The creation of these novel media will significantly add to the scientific community’s ability to better model CF infection dynamics in vitro preclinical models. Including healthy lung and sinus media will no doubt serve as a useful control for these experiments than LB or M9. Having accurate models for upper airway infection is also key in understanding the intermittent colonization and persistence of P. aeruginosa during initial colonization and antibiotic treatment.\nThe various components tested by the authors for the development of these novel media were: Lysozyme, Lactoferrin, Spermidine, Spermine, Putrescine, Mucin, eDNA, Albumin, CaCl2, MgCl2, FeCl2, CuCl2, ZnCl2, NaCl, Sialic acid, Bile, N-acetyl glucosamine, Glucose, Succinate, Galactose, L-Methionine, L-Phenylalanine, LProline, L-Serine, L-Threonine, L-Tryptophan, L-Valine, L-Ornithine, L-Tyrosine, L(+)-Asparagine monohydrate, L-Alanine, L-Arginine, L(+)-Aspartic acid, L-Cysteine, LGlutamine, L-Glycine, L-Histidine, L-Isoleucine, L-Leucine, and L-Lysine. The authors chose the PAO1 and LESB65 as representative laboratory and CF-adapted strains of P. aeruginosa in their tested conditions. The authors first grew these strains in M9 media supplemented with each chemical component independently at three different concentrations. These concentrations were selected to most closely reflect the physiological concentration of each compound within each niche, based upon cited references in the literature or experimental testing of metabolites. Upon confirmation that these strains could grow sufficiently at the selected concentration of each metabolite, the authors then pooled the components at the appropriate ratios to yield four novel respiratory media: Healthy Sinus Media (HSM), Healthy Lung Media (HLM), Cystic Fibrosis Sinus Media (CFSM), and Cystic Fibrosis Lung Media (CFLM).\nThe authors tested the viability and relevance of these media as preclinical models for human respiratory infections by assessing the: i) growth and biofilm formation of strains cultured continuously for 72 hours; ii) viscosity and shear rate in the media pre- and post-biofilm formation; iii) the stability of media after storage at 4° C for 40 days; iv) transcriptional changes of infection-relevant genes; and v) changes in colony morphology of strains evolved for 40 days; of PAO1 and LESB65 in each medium.\nThe growth rate experiments show that PAO1 and LESB65 could be maintained for at least 72 hours, with similar dynamics to those previously published for P. aeruginosa in SCFM (authors may include the publications that used these media to study various aspects of P. aeruginosa infection). The crystal violet assays, which included growth in M9 and Luria-Bertani (LB) as additional comparators against the newly described media, show niche, disease state, and strain-specific differences in surface-attached and free-floating biofilm formation, highlighting the importance of using clinically-relevant models for estimating biofilm formation. Although the aggregate and aggregate formation are not tested here, authors can discuss those future assessments for these media.\nThe authors also show that the viscosity and shear rate measurements of their proposed media agree with those published previously for CF and healthy human sputum. The authors also show that each media could support the same level of biofilm formation in PAO1 after 0, 10, 20, and 30 days of storage at 4° C, implying that the media remains stable for this duration. qPCR analysis shows that expression of the four tested virulence genes—algU, mexB, PA2911, and PA2382—significantly increased in all four novel media compared to their transcription levels in LB and additionally were comparable to transcription levels of the same strains grown in SCFM2. Long-term culturing of PAO1 in these media shows the evolution of a wrinkly colony morphotype, a potential redox-driven adaptation to maximize oxygen availability in biofilms, and morphological adaptations specific to CF media.\nSome minor comments:\nThe authors determined these novel media's viability over 30 days by measuring PAO1 biofilm formation at 0, 10, 20, and 30 days. While it is nice to show that the media sustain similar levels of biofilm growth regardless of whether the media is freshly prepared, this might not be the best indicator of media stability over long periods. Instead, it would have been nice to see the authors conduct LCMS analyses to determine the stability of each of the chemical components at 0 and 30 days and detect if any possible degradation had occurred. Also, it is important to determine whether the growth of P. aeruginosa in the stored media displays any altered metabolism, physiology, or spatial organization.\nAdditionally, instead of hand-picking a short list of four genes, it would have greatly strengthened the supporting case for these novel media by RNA-seq analysis. For example, comparing the entire transcriptional profile of P. aeruginosa grown in new media (fresh and stored at 4° C) to the transcriptional profile of cells grown in SCFM2 and in vivo samples would confirm the similarity of these preclinical models to the infection and healthy environments.\nI also understand that these experiments can be addressed in the future and are out of the scope of this study, which aimed to introduce these novel media for upper and lower respiratory tracts; however, I recommend that authors address this in the discussion section. I recommend authors include these studies and discuss the key benefits of using the novel HSM, CFSM, HLM, and CFLM media over the other versions available.\nIn the section titled “Airway mimicking media is stable for at least 30 days at 4° C,” the wording of the first sentence leads to some confusion. At first glance, it appears to imply that PAO1 was cultured for 30 days, although the authors meant to say that PAO1 was cultured in media that had been stored for 30 days. The authors may want to re-write this sentence to avoid confusion.\nIn Tables 3, 8, and 12, please use a consistent format for the concentration units μg/L or μg/l.\nIn Figure 5, the legend below the chart is redundant, as it shows the same data as the labels on the X-axis. I would recommend removing this legend to simplify the readers' view.\nIn Figure 6, standard deviation bars and significance labels would help the reader understand which relationships are significant without having to reference the text.\nThere are studies on the evolution of P. aeruginosa in TSB, SCFM, and SCFM2 by Schick A, Kassen R (2018)1, Azimi et al., 20202, and Flynn et al., 20163, showing the emergence of various morphotypes in the populations that authors may want to include.\nThe emergence of the observed morphotypes and phenotypes in this study is similar to what is observed as a result of evolving P. aeruginosa in other preclinical infection models are not unique to novel developed media. I suggest that authors include previous studies, discussing how future studies of genetic and phenotypic adaptation to the developed media are required. Thus, future analysis can confirm the unique adaptive traits observed in this study, similar to upper and lower CF respiratory infections.\nIn Table 14, I believe the title is somewhat of a misnomer. The table shows when each morphotype appears and whether it is maintained in the populations. I would consider re-titling this table to reflect the prevalence of each morphotype over the evolution period.\n\nAre a suitable application and appropriate end-users identified? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate? Yes\n\nAre the 3Rs implications of the work described accurately? Yes\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": "9031",
"date": "30 Nov 2022",
"name": "DILEM RUHLUEL",
"role": "Author Response",
"response": "Thank you for the positive comments regarding the manuscript and for your helpful suggestions for improvements and discussion points. We have addressed each point raised, below and have submitted an updated manuscript version in which the changes have been made. The authors determined these novel media's viability over 30 days by measuring PAO1 biofilm formation at 0, 10, 20, and 30 days. While it is nice to show that the media sustain similar levels of biofilm growth regardless of whether the media is freshly prepared, this might not be the best indicator of media stability over long periods. Instead, it would have been nice to see the authors conduct LCMS analyses to determine the stability of each of the chemical components at 0 and 30 days and detect if any possible degradation had occurred. Also, it is important to determine whether the growth of P. aeruginosa in the stored media displays any altered metabolism, physiology, or spatial organization. We agree both that LCMS would offer a more complete determination of chemical stability and that investigation of bacterial metabolism, physiology and spatial organisation would be desirable. We have added a sentence to the discussion, highlighting the need for further studies into media stability. These studies are underway in our lab. Additionally, instead of hand-picking a short list of four genes, it would have greatly strengthened the supporting case for these novel media by RNA-seq analysis. For example, comparing the entire transcriptional profile of P. aeruginosa grown in new media (fresh and stored at 4° C) to the transcriptional profile of cells grown in SCFM2 and in vivo samples would confirm the similarity of these preclinical models to the infection and healthy environments. I also understand that these experiments can be addressed in the future and are out of the scope of this study, which aimed to introduce these novel media for upper and lower respiratory tracts; however, I recommend that authors address this in the discussion section. I recommend authors include these studies and discuss the key benefits of using the novel HSM, CFSM, HLM, and CFLM media over the other versions available. We completely agree and these studies have already begun. The large RNAseq dataset will require careful analysis. We feel that the space and word count needed to describe those data fully would make the current manuscript overly lengthy. As the reviewers suggest, we have instead mentioned the need for these investigations in the discussion. In the section titled “Airway mimicking media is stable for at least 30 days at 4° C,” the wording of the first sentence leads to some confusion. At first glance, it appears to imply that PAO1 was cultured for 30 days, although the authors meant to say that PAO1 was cultured in media that had been stored for 30 days. The authors may want to re-write this sentence to avoid confusion. Sorry for this confusion, we have now reworded the sentence. In Tables 3, 8, and 12, please use a consistent format for the concentration units μg/L or μg/l. We have now used μg/l throughout. In Figure 5, the legend below the chart is redundant, as it shows the same data as the labels on the X-axis. I would recommend removing this legend to simplify the readers' view. This figure has been reworked and the legend removed. In Figure 6, standard deviation bars and significance labels would help the reader understand which relationships are significant without having to reference the text. Standard deviation bars and significance labels have now been added to these graphs and we have described ANOVA summary statistics in the main text. There are studies on the evolution of P. aeruginosa in TSB, SCFM, and SCFM2 by Schick A, Kassen R (2018)1, Azimi et al., 20202, and Flynn et al., 20163, showing the emergence of various morphotypes in the populations that authors may want to include. Thank you for highlighting this. Reference to these studies has been added to our discussion. The emergence of the observed morphotypes and phenotypes in this study is similar to what is observed as a result of evolving P. aeruginosa in other preclinical infection models are not unique to novel developed media. I suggest that authors include previous studies, discussing how future studies of genetic and phenotypic adaptation to the developed media are required. Thus, future analysis can confirm the unique adaptive traits observed in this study, similar to upper and lower CF respiratory infections. Thank you for this suggestion. We have added the three references mentioned in the previous comment, alongside some discussion of the need to deconvolute those phenotypes that are adaptive for different respiratory environments from those that are merely adaptive to different modes of growth. In Table 14, I believe the title is somewhat of a misnomer. The table shows when each morphotype appears and whether it is maintained in the populations. I would consider re-titling this table to reflect the prevalence of each morphotype over the evolution period. We have re-titled this table. Unfortunately, these data were captured as presence/absence for each morphotype, at each time point. We don’t, therefore, have information on relative or absolute morphotype abundance over time."
}
]
}
] | 1
|
https://f1000research.com/articles/11-1007
|
https://f1000research.com/articles/11-1407/v1
|
30 Nov 22
|
{
"type": "Case Report",
"title": "Case Report: Spontaneous remission of early T-cell precursor acute lymphoblastic leukemia",
"authors": [
"Amira Cherif",
"Veronique Saada",
"Amina Bouatay",
"Veronique Saada",
"Amina Bouatay"
],
"abstract": "Background: Spontaneous remission (SR) has been reported in different hematological malignancies. It has been observed in adult T-cell lymphoma, chronic lymphocytic leukemia (CLL) and myelodysplastic syndrome (MDS). It is generally associated with recovery from an infectious or immunological process, and more recently possibly with clonal hematopoiesis. Case: We reviewed the literature and reported a new case of a 40 year-old man with a morphologic and cytogenetic diagnosis of early T-cell precursor acute lymphoblastic leukemia (ALL) associated with an appendicular abscess. During his hospitalization and surgical management of his appendicitis, we noted SR of the rate of blast cells until cytological and cytogenetic remission of his ALL but unfortunately it did not last too long, moreover our patient relapsed after nine months, received intensive chemotherapy, underwent a placental blood allograft but relapsed again and died. Conclusions: In contrast to SR in other types of cancer, all documented cases of SR in ALL were only transient, so is there a need for early cytotoxic therapy in SR in ALL to delay relapse?",
"keywords": [
"spontaneous remission",
"acute lymphoblastic leukemia",
"lymphoblastic lymphoma",
"relapse"
],
"content": "Introduction\n\nThe partial or complete, temporary or definitive disappearance of a malignant pathology without any specific treatment is referred to as spontaneous remission (SR) or regression of a cancer. For solid tumors, the expression “spontaneous regression” is used, whereas for leukemias, we speak of “spontaneous remission”.1,2 SR has been reported in various hematological malignancies, for example: in adult T-cell lymphoma,3 in chronic lymphocytic leukemia (CLL),4 in myelodysplastic syndrome (MDS),5 in acute myeloid leukemia (AML), rarely in acute lymphoblastic leukemia (ALL) in children and exceptionally in adults.2,6,7 SRs are often related to infections or other immune stimulators, resulting in potent immune activation playing a crucial role in controlling the leukemic clone.8,9 Nevertheless, the underlying mechanism of this phenomenon is not clearly described.\n\nIn this article, we report the case of a patient consulting for appendicular syndrome who was found to have blastic invasion during the preoperative workup, which regressed spontaneously during his hospitalization.\n\n\nCase presentation\n\nWe report the case of a 40 year old man from Martinique with no past medical or surgical history who presented to the Emergency Department complaining of abdominal pain and fever. On abdominal ultrasound, an appendicular abscess was noted and on the count blood cells (CBC) the diagnosis of acute leukemia was made with leucopenia (white blood cells (WBCs) = 1.6×109/L) and 12% of circulating blast cells. His bone marrow aspiration was hypercellular with infiltration by 65% of undifferentiated blast cells (Figure 1). The patient did not undergo surgery due to the discovery of leukemia and the risks associated with the surgery in the context of anticipated aplasia, so the patient was put on first-line antibiotic therapy: cefepime (2 g × 2/day) and ciprofloxacin (750 mg × 2/day) for 7 days and transferred to the Gustave Roussy Institute (GRI) on December 14 for the management of his leukemia.\n\nThe bone marrow aspirate smears show hypercellular bone marrow (A, B) with infiltration by 65% of undifferentiated blast cells (C) (100×, May-Grünwald Giemsa stain). ALL (acute lymphoblastic leukemia).\n\nOn the hematological level, the evolution at the GRI was marked by a regression of the rate of circulating blasts and improvement of the leucopenia.\n\nThe CBC showed anemia (Hb 10.5g/dL), leucopenia (WBC 1.9*109/L) and normal rate of platelet (425*109/L). The differential leukocytic count showed neutropenia (0.8×109/L) and 2% of blast cells detected in a blood film. Repeat bone marrow aspiration revealed a marrow with moderate richness characterized by a hyperplasia of the erythroblastic compartment (55% among medullary figurative elements) associated with moderate signs of dyserythropoiesis and an excess of undifferentiated blasts (12%). The blast cells were small with agranular rim of cytoplasm and very high nucleocytoplasmic ratio. The nucleus has fine chromatin with one to two nucleoli (Figure 2).\n\nThe bone marrow aspirate smears show hyperplasia of the erythroblastic compartment with 55% erythroblast (A, B). Undifferentiated blast cells comprise 12% of bone marrow cellularity (C) with moderate signs of dyserythropoiesis (100x, May-Grünwald Giemsa stain).\n\nFlow cytometry of the bone marrow aspirate revealed blasts positive for CD45 dim (15%), immaturity marker: CD34+ (85%), CD117+ (22%), HLADR+ (40%) and myeloid marker: CD33+ (75%), cCD13+ (68%), CD11b (61%) and expressing lymphoid T marker: CD5+ (67%), CD7+ (85%), cCD3+ (74%) (Figure 3), suggesting the possibility of an early T-cell acute lymphoblastic leukemia (ETP-ALL) according to the 2016 WHO acute leukemia classification.10 We also note a hyperplastic erythroblastic contingent: CD36+ (47%), Glycophorin A+ (26%), in agreement with the cytology. Molecular cytogenetic technique, such as Fluorescent in situ hybridization (FISH) showed a trisomy 8. Three days later a bone marrow aspiration was released and ETP-ALL was confirmed.\n\nALL (acute lymphoblastic leukemia); CD (cluster of differentiation); PE (phycoerythrin).\n\nAt GRI, the diagnosis of appendicitis without signs of complication was confirmed. After one week of observation, abdominal pains persisted and the appendicular syndrome did not regress in spite of an antibiotic therapy, requiring surgical intervention on December 20. The patient underwent a laparoscopic appendectomy, without postoperative anomalies.\n\nAt the same time, we noted a progressive normalization of the hemogram, only the anemia remained. Indeed, on December 31, CBC showed Hb 10.6 g/dL (anemia) and normal rate of WBC 4.2×109/L and platelet (432×109/L). The myelogram showed a rich, polymorphic, slightly erythroblastic marrow. The blastic infiltration seemed less important, evaluated at 4%. Therefore, no cytological aspect of acute leukemia was noted on this sample.\n\nTwo weeks later, CBC showed Hb 11.3g/dL, WBC 6.9×109/L and the differential leukocytic count showed 4.4×109/L neutrophils, 1.4×09/L lymphocytes, 0.9×109/L monocytes and 0.2×109/L eosinophils with no blast cells detected in a blood film. A bone marrow aspiration revealed a stable blastosis compared to the previous myelogram with 5% blasts and cytogenetic analysis also confirmed a cytogenetic remission (Figure 4).\n\nBone marrow aspirate smears show maturing hematopoiesis with no increased blasts (A,B) (100×, May-Grünwald Giemsa stain).\n\nIn view of the patient's clinical improvement and the SR of his acute leukemia, the patient was discharged from the hospital. He underwent a human leukocyte antigen (HLA) typing in order to search for a donor on file in case of the need for a marrow transplant in the event of relapse.\n\nNine months from first documented SR of the ALL, the patient relapsed. His bone marrow aspiration revealed a poor bone marrow with hyperplasia of the erythroblastic compartment (43% among medullary figurative elements) associated with moderate signs of dyserythropoiesis and an excess of undifferentiated blasts (50%) (Figure 5). Flow cytometry of the bone marrow aspirate revealed an early T-cell acute lymphoblastic leukemia (Figure 6). So, he received intensive chemotherapy according to the following protocol: daunorubicin (60 mg/m2 at day (D)1, D2, D3, D8 and D9) + cytarabine (30 mg/m2/D at D2 and D6) + aracytin (1,000 mg/m2/D at D1 and D6). Five months later, he underwent a placental blood allograft after conditioning with cyclophosphamide (50 mg/kg at D6), fludarabine (200 mg/m2/D at D6 and D2), total body irradiation (2 Gy at D1) but relapsed in the following year. Administration of two courses of cytarabine (1 g/m2 at D1 to D5) + clofarabine (30 mg/m2 at D2 to D6) was initiated but the patient remained very cytopenic without blast cells for another month to relapse again and died after two weeks.\n\nThe bone marrow aspirate smears show a poor bone marrow (A,B) with hyperplasia of the erythroblastic compartment (43%) and an excess of undifferentiated blasts (50%) (C) (100×, May-Grünwald Giemsa stain).\n\nCD (cluster of differentiation); PE (phycoerythrin).\n\n\nDiscussion\n\nAcute leukemia with spontaneous remission has been reported since 1878.1 A PubMed (RRID:SCR_004846) search using the terms “acute leukemia”, “remission”, “spontaneous”, including only articles written in English excluding children, yielded a total of 49 articles that were reviewed and 60 cases reported of acute leukemia with SR. A total of 76% of these patients had an associated infection and 45% of them received a blood product transfusion. Less common associations were growth colony stimulating factor (G-CSF), steroids, hydroxyurea, termination of pregnancy, gonadotropin releasing hormone (GnRH), tumor lysis syndrome, discontinuation of lenalidomide, and Henoch-Schönlein purpura. A total of 9% had no identifiable association.11\n\nSR in acute lymphoblastic leukemia is a rare phenomenon. Among the 60 cases studied, only eight patients had ALL with SR. Table 1 resumes our case and all cases of ALL with SR reported in the literature. ALL with SR was observed in both male and female patients with equal prevalence and a median age of 32.25 years. We note that the duration of remission is very variable and can be few days (14 days) to several years (>8 years).\n\n* Had SR but also received therapy.\n\nHere, we present the case of early T-cell precursor ALL diagnosed on cytological criteria and on Flow Cytometric Immunophenotyping of blasts in bone marrow. He did not start chemotherapy since a progressive decrease of the blast rate was noticed. During follow up and monitoring, the patient showed CBC recovery and the blastic infiltration seemed less important, evaluated at 4%. Therefore, no cytological aspect of acute leukemia was noted and cytogenetic remission was confirmed. Although the mechanisms of SR are not clear, they include infections.2 Indeed, an immune-mediated cause secondary to a previous severe infection is one of the most common proposals12 that may contribute to leukemia remission through excessive activation and production of pro-inflammatory cytokines, such as tumor necrosis factor (TNF) and interleukin 2 (IL-2). These cytokines, all of which have an anti-leukemic effect, increase the activity of T cells, macrophages and natural killer (NK) cells.13,14 A case of SR of adult T-cell leukemia/lymphoma (ATL) associated to pneumonia was reported.3\n\nOther possible mechanisms of SR are hormonal factors, tumor necrosis, inhibition of angiogenesis, apoptosis, adverse effects of blood transfusions and elimination of carcinogenic substances. Especially the association between transfusion of different blood components and SR was reported.6 Indeed, a possible anti-leukemic effect could be observed especially in patients who have been treated with non-irradiated blood products. This effect may stimulate remission mediated by transfusion-associated graft-versus-host disease (TA-GVHD) and graft-versus-host leukemia (GVL) reaction.\n\nOur case went through SR for nine months. Indeed, ETP-ALL is a different subtype of T-cell lymphoblastic leukemia, first identified in 2009, due to its unique immunophenotypic and genomic profile. The prognosis with these patients was poor in previous studies because they were susceptible to induction failure, with more frequent relapse/refracture disease.15 This may explain the relatively short time to relapse. Reports of spontaneous remissions of ALL have shown periods of temporary remission varying in duration from 14 days to 8 years.16,17 It should be noted that the longest periods of remission are generally with patients who had SR but also received therapy.\n\n\nConclusions\n\nSR of ALL is an uncommon and transient phenomenon9 that is rarely described in the literature. Its mechanisms are not well elucidated; however, it is necessary to better understand the processes underlying SR as this could lead to new therapies for ALL. Also, there is a relevant question that requires careful consideration: “Is it necessary to implement early cytotoxic therapy during spontaneous remission in ALL, since the remissions are transient?”\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\nEisa N, El-Ashwah S, Denewer M, et al.: Spontaneous remission in acute lymphoblastic leukemia: a case report and review of the literature. Hematol. Transf. Inter. J. 2017; 4(4): 100–101.\n\nYoruk A, Erguven M, Celiker E, et al.: Spontaneous remission of acute lymphoblastic leukemia with mediastinal mass. Pediatr. Hematol. Oncol. 2008; 25(3): 181–186. PubMed Abstract | Publisher Full Text\n\nTakezako Y, Kanda Y, Arai C, et al.: Spontaneous remission in acute type adult T-cell leukemia/lymphoma. Leuk. Lymphoma. 2000; 39(1-2): 217–222. PubMed Abstract | Publisher Full Text\n\nDel Giudice I, Chiaretti S, Tavolaro S, et al.: Spontaneous regression of chronic lymphocytic leukemia: clinical and biologic features of 9 cases. Blood. 2009; 114(3): 638–646. PubMed Abstract | Publisher Full Text\n\nPetti MC, Latagliata R, Breccia M, et al.: Spontaneous remission in adult patients with de novo myelodysplastic syndrome: a possible event. Haematologica. 2001; 86(12): 1277–1280. PubMed Abstract\n\nKaźmierczak M, Szczepaniak A, Czyż A, et al.: Spontaneous hematological remission of acute myeloid leukemia. Contemp. Oncol. (Pozn). 2014; 18(1): 67–69. PubMed Abstract | Publisher Full Text\n\nPurohit A, Aggarwal M, Kumar S, et al.: Spontaneous remission of adult acute lymphoblastic leukemia: a very rare event. Indian J. Hematol. Blood Transfus. 2015; 31(1): 159–160. PubMed Abstract | Publisher Full Text\n\nRashidi A, Fisher SI: Spontaneous remission of acute myeloid leukemia. Leuk. Lymphoma. 2015; 56: 1727–1734. Publisher Full Text\n\nMartínez-Díez Y, Franganillo-Suárez A, Salgado-Sánchez R, et al.: Spontaneous remission of acute myeloid leukemia: A case report. Medicina. 2022; 58(7): 921. PubMed Abstract | Publisher Full Text\n\nArber DA, Orazi A, Hasserjian R, et al.: The 2016 revision to the World Health Organization (WHO) classification of myeloid neoplasms and acute leukemia. Blood. 2016; 127: 2391–2405. PubMed Abstract | Publisher Full Text\n\nBradley T, Zuquello RA, Aguirre LE, et al.: Spontaneous remission of acute myeloid leukemia with NF1 alteration. Leuk. Res. Rep. 2020; 13: 100204. PubMed Abstract | Publisher Full Text\n\nHelbig D, Quesada AE, Xiao W, et al.: Spontaneous remission in a patient with acute myeloid leukemia leading to undetectable minimal residual disease. J. Hematol. 2020; 9(1-2): 18–22. PubMed Abstract | Publisher Full Text\n\nMusto P, D’Arena G, Melillo L, et al.: Spontaneous remission in acute myeloid leukemia: a role of endogenous production of tumor necrosis factor and interleukin-2? Br. J. Haematol. 1994; 87(4): 879–880. Publisher Full Text\n\nJimemez C, Ribera JM, Abad E, et al.: increased serum tumor necrosis factor during transient remission in acute leukemia. Lancet. 1993; 341(8860): 1600. Publisher Full Text\n\nSin CF, Marcus Man PH: Early T-Cell Precursor Acute Lymphoblastic Leukemia: Diagnosis, Updates in Molecular Pathogenesis, Management, and Novel Therapies. Front. Oncol. 2021; 11: 750789. PubMed Abstract | Publisher Full Text\n\nChen RL, Chuang SS: Transient spontaneous remission after tumor lysis syndrome triggered by a severe pulmonary infection in an adolescent boy with acute lymphoblastic leukemia. J. Pediatr. Hematol. Oncol. 2009; 31(1): 76–79. PubMed Abstract | Publisher Full Text\n\nLüke F, Harrer DC, Hahn J, et al.: Continous complete remission in two patients with acute lymphoblastic leukemia and severe fungal infection following short-term, dose reduced chemotherapy. Front. Pharmacol. 2021; 21: 599552.\n\nHöres T, Wendelin K, Schaeffer-Eckart K: spontaneous remission of acute lymphoblastic leukemia: A case report. Oncol. Lett. 2018; 15: 115–120. PubMed Abstract | Publisher Full Text"
}
|
[
{
"id": "205124",
"date": "27 Sep 2023",
"name": "Rosana Pelayo",
"expertise": [
"Reviewer Expertise Childhood leukemia"
],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe study submitted by Cherif et al. shows an interesting case of apparent spontaneous remission in a patient diagnosed with ETP-ALL harboring trisomy 8 in blasts, who was treated with antibiotics for abdominal infection. After nine months without evidence of leukemia or trisomy 8, the patient suffered relapse and ultimately died.\nThis reviewer suggests that a few points be addressed for the sake of clarity and quality:\nPlease comment on treatment with nelarabine for this type of ALL, since it is currently one of the recommended therapy 1,2.\nAnother critical point is the extramedullary infiltration frequently associated with ETP-ALL. The status of the cerebrospinal fluid is not found in the summary, nor is the pathology report of the removed appendix, which is essential to discuss. Despite the spontaneous remission of the bone marrow, it cannot be ruled out the possibility of an extramedullary reservoir allowing a disease persistence while bone marrow remission 3,4\nIt is suggested to avoid mentioning in the summary registered brand names (aracytin).\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": []
}
] | 1
|
https://f1000research.com/articles/11-1407
|
https://f1000research.com/articles/11-1406/v1
|
30 Nov 22
|
{
"type": "Research Article",
"title": "The effect of obesity on the outcome of total knee replacement (TKR)",
"authors": [
"Abdullah AlOmran"
],
"abstract": "Background: The objective of this study was to look for the outcome of TKR in obese individuals. Methods: The data of all patients who had undergone primary TKR between 2005 and 2015 were collected from the QuadruMed Patient Care System and crosschecked with the operating room. The biometric data of age, gender, weight and postoperative follow up, any complications were collected from the medical charts. Patients were classified as per the BMI (Body Mass Index). BMI is classified as per the WHO classification of 2000. Results: Two hundred and four patients fulfilled the criteria for inclusion. The demographic data is given in Table 2. The average age 59.8±9.3 years. Right side was operated in 103 and left in 101 patients. The average BMI was 35.88±7.47 (Range 21.95-54.78); mean duration of surgery was 163.37±29.9 (Range 88-250) minutes. The range of follow up was 5-14 years with the average of 7.49±2.56 years. There were 18 (8.8%) complications. One hundred and three (50.5%) of the patients belonged to Class II Obesity (BMI ≥36Kg/M2). Between class II obesity and Class III obesity (BMI ≥41Kg/M2, the later had 94.4% of the complications. The level of satisfaction was similar in all the groups at 80%. High BMI and longer duration of surgery were important and statistically significant risk factors (p <0.0001 and <0.009) for complications during TKA. Post operative data on VAS, Modified Knee Scoring Score and WOMAC grading, showing low scores of all the assessment in the patients with higher BMI. Conclusions: Our analysis shows that our patients with Class III obesity had over 90% of the complications and we believe that patients in this BMI range should seek ways to reduce their body weight to avoid complications and long-term morbidity.",
"keywords": [
"Osteoarthritis of knee",
"Total Knee Arthroplasty",
"Obesity",
"Complications",
"Morbidity."
],
"content": "Editorial note\n\nEditorial Note (29th June 2023): Since publication, concerns have been raised to the Editorial Team regarding the ethical approval and the dataset used in this research. The Editorial Team requested the raw underlying data and ethical approval details from the authors on 22nd March 2023. The authors have not provided the raw underlying data despite repeated requests and issues with the ethical approval remain. The Editorial Team contacted the authors' institution on 22nd June 2023 in order to seek further information on this matter. The Editorial Team will update this Editorial Note as the situation progresses. Peer review activity has been suspended for this article until an explanation has been provided by the authors or their institution.\n\n\nIntroduction\n\nOsteoarthritis of the Knee (OAK) is common and debilitating degenerative disease, which pins the patients down to the wheelchairs in the later part and requires a Total Knee Replacement (TKR) for relief of pain and to increase mobility. It has been suggested that age, sex and body weight influence the severity of the disease. The prevalence of OA of the knee is unknown but recently it was an estimated that 12.5% of the general population aged ≥45 years suffers from OA of the knee.1 Among the ethnic Saudi Arabian population OA of the knee was reported to be between 1.19 to 3.5%,2,3 but in a decade and half the prevalence of clinical OA of the knee jumped to 13%4 and radiological OA of knee to 53% in males and 60.9% in females.5 Total knee replacement now is an important and successful procedure which can relieve the patients of pain and suffering and limit the complications of immobility in the advanced age. It has been reported that over a million TKRs are performed in the world and the numbers are increasing in many countries.6\n\nComplications after TKR can occur are not uncommon and there is a long list which can occur after routine surgery but obesity is known to be an important risk factor in increasing the complications.7–9 There is enough evidence that complications post TKR in obese patients are higher than those with the normal weight patients, to the extent that in United Kingdom obese patients are being denied surgery before they loose weight prior to TKR.10,11 In spite of this number of morbidly obese patients with OAK are getting their joints replaced.12 Studies have also shown that there is no additional risk of complications in obese and morbid obese patients.13,14\n\nAn extensive review of literature did not reveal any reported studies on this aspect in Saudi Arabia nor the Arab world, even though WHO reports 30% of the population in the region is obese and overweight. Hence we decided to retrospectively study the effect of obesity of the outcome of Total Knee Replacement performed at the King Fahd Hospital of the University at AlKhobar.\n\n\nMethods\n\nThe study was carried out after receiving the approval by the Institutional Review Board of Imam AbdulRahman Bin Faisal University, Dammam and informed written consent was taken from all the patients prior to surgery and with approval of publishing the data without revealing their names. STROBE guidelines were followed in reporting of the observational studies. The data of all patients who had undergone primary TKR between 2005 and 2015 were collected from the QuadruMed Patient Care System and crosschecked with the operating room. The biometric data of age, gender, weight, and postoperative follow up, any complications will be collected from the medical charts. Patients were classified as per the BMI (Body Mass Index). BMI is classified as per the WHO classification of 200015 (Table 1). Patients were classified as per the WHO classification and grouped accordingly based on the BMI. Only patients who came for final follow up were included for analysis. Gender differences were not taken into consideration for the design of the study nor for analysis of results as only BMI was considered as a factor for complications. At the final follow up Knee society score (KSS) will be filled up. The patients’ the SF-36 questionnaire (version 1) and knee stability, ROM, pain, and functional ability will be assessed and the WOMAC grading were calculated. Only those patients who came for the final follow up and had at least of five years of follow up were included for the analysis. Post hoc power analysis for comparing the four groups was done to and found the sample size was adequate for the comparison. Patients who completed the follow-up period and came for the final examination were compared the t-test. For the statistical analysis, the SPSS Inc. ver 25 was used. All p-values < 0.05 were considered statistically significant.\n\n\nResults\n\nTwo hundred and four patients fulfilled the criteria for inclusion. The demographic data is given in Table 2. The average age 59.8±9.3 years. Right side was operated in 103 and left in 101 patients. The average BMI was 35.88±7.47 (Range 21.95-54.78); mean duration of surgery was 163.37±29.9 (Range 88-250) minutes. The range of follow up was 5-14 years with the average of 7.49±2.56 years. There were 18 (8.8%) complications. Table 3 shows the comparison of outcome based on BMI. One hundred and three (50.5%) of the patients belonged to Class II Obesity (BMI ≥36 kg/m2). Complications related to the surgery are given in Table 4. Between class II obesity and Class III obesity (BMI ≥41 kg/m2, the later had 94.4% of the complications. The level of satisfaction was similar in all the groups at 80%. High BMI and longer duration of surgery were important and statistically significant risk factors (p<0.0001 and <0.009) for complications during TKA (Table 5). Table 6 gives the post-operative data on VAS, Modified Knee Scoring Score and WOMAC grading, showing low scores of all the assessment in the patients with higher BMI.\n\n\nDiscussion\n\nOur study shows that patients with higher BMI have higher risk to develop complications after TKR. The second important risk factor we have observed was the duration of surgery. The longer the duration of surgery higher was the risk. There has been a conflicting opinion with regard to the influence of obesity on the outcome of the TKR. DeMik et al (2021)16 concluded that patients with BMI ≥ 40 kg/m2 suffer from higher risk of superficial and deep infections when compared to patients who are non-morbidly obese. Earlier McElroy et al (2013)17 reported that incidence of complications in obese and morbidly obese patients the observed complications were 15, and 22%. In our study the complications were 18.8% in the two groups. Contrary to this belief, reports also indicates that obese patients undergoing joint arthroplasty do as good as normal weight patients.18,19 Recent studies concluded that weight of the patients did not effect the outcome in clinical and functional improvement.20–22 Our study has convincingly showed otherwise. Nearly all patients with Class II and III obesity had complication. This study further supports the policy of United Kingdom, which made mandatory to reduce weight before joint replacement surgery.10,11 In our study we did not find and difference of complications between male and female patients, but the high BMI was the driving force for increased complications. We concur with the other studies on negative impact of gender on complications.23,24\n\nDuration of surgery influences complications in any surgery and joint arthroplasty should be an exception. Yasunaga et al (2009)25 suggested that with the average operating time was 127 ± 47 min; they had postoperative complications of 9.8%. A decade down the line Ravi et al (2019)26 reported in their review that the safe cut off point of 100 minutes to be ideal duration and anytime longer than that will carry a risk of deep infection. In our study the average time of the surgery was 163 minutes but in obese patients the average time was ≥20 minutes which could have added to the increased complications.\n\nThe prevalence of obesity is increasing in the World and Saudi Arabia is not behind.\n\nAl-Raddadi et al (2019)27 reported that the pervasiveness of obesity in the Saudi Arabian population persists high and preventing measures are not working. This makes the incidence of OAK to become higher as overweight women carry four times and men five times the risk of developing OAK and the association of obesity and OAK is well established.28 More and more obese and morbidly obese patients are being offered TKR and many of these patients ending up with complications, revisions and extortionate levels of morbidity.\n\n\nConclusions\n\nOur study indicates that obese and morbidly obese patients undergoing TKR carry a high risk of complications post TKR and these patients should be counseled to reduce weight before they envisage joint replacement to avoid unnecessary complications.",
"appendix": "Data availability\n\nFigshare. The effect of Obesity on the Outcome of Total Knee Replacement (TKR), DOI: https://doi.org/10.6084/m9.figshare.21295869. 29\n\nThis project contains the following data:\n\n- Effect of obesity on Total Knee arthroplasty\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\nJordan K: Croft P The prevalence and history of knee osteoarthritis in general practice: a case–control study. Fam. Pract. 2005; 22(1): 103–108.\n\nSadat-Ali M, Al-Gindan Y, Al-Mousa M, et al.: Osteoarthritis of the knee among Saudi Arabian security forces personnel. Mil. Med. 1996 Feb; 161(2): 105–107. PubMed Abstract | Publisher Full Text\n\nAhlberg A, Linder B, Binhemd TA: Osteoarthritis of the hip and knee in Saudi Arabia. Int. Orthop. 1990; 14(1): 29–30. PubMed Abstract | Publisher Full Text\n\nAl-Arfaj AS, Alballa SR, Al-Saleh SS, et al.: Knee osteoarthritis in Al-Qaseem. Saudi Arabia Saudi Med. J. 2003 Mar; 24(3): 291–293. PubMed Abstract\n\nAl-Arfaj A, Al-Boukai AA: Prevalence of radiographic knee osteoarthritis in Saudi Arabia. Clin. Rheumatol. 2002; May; 21(2): 142–145. PubMed Abstract | Publisher Full Text\n\nKurtz SM, Lau E, Ong K, et al.: Future young patient demand for primary and revision joint replacement: national projections from 2010 to 2030. Clin. Orthop. Relat. Res. 2009; 467: 2606–2612. PubMed Abstract | Publisher Full Text\n\nHawker GA, Croxford R, Bierman AS, et al.: Lipscombe LL All-cause mortality and serious cardiovascular events in people with hip and knee osteoarthritis: a population based cohort study. PLoS One. 2014; 9: e91286. PubMed Abstract | Publisher Full Text\n\nConnelly AE, Tucker AJ, Kott LS, et al.: Modifiable lifestyle factors are associated with lower pain levels in adults with knee osteoarthritis. Pain Res. Manag. 2015; 20: 241–248. PubMed Abstract | Publisher Full Text | Free Full Text\n\nCarter J, Springer B, Curtin B: Early Complications of Revision Total Knee Arthroplasty in Morbidly Obese Patients. Clin. Surg. 2016; 1: 1056.\n\nNHS Warwickshire: Commissioning Policy Statement: Referral and Surgical Threshold Criteria for Elective Primary Hip Replacement Surgery.2011. Reference Source\n\nNHS Devon, NHS Plymouth, Torbay Care Trust. Peninsula Commissioning Priorities Group: Commissioning Decision: Hip and Knee Replacement Surgery in Obese Patients (Those with a Body Mass Index of 30 or Greater).2011. Reference Source\n\nFehring TK, Odum SM, Griffin WL, et al.: The obesity epidemic: its effect on total joint arthroplasty. J. Arthroplast. 2007; 22: 71–76. Publisher Full Text\n\nBieger R, Kappe T, Jung S, et al.: Does the body mass index influence the results of revision total knee arthroplasty? Z. Orthop. Unfall. 2013; 151: 226–230. PubMed Abstract | Publisher Full Text\n\nRajan TS, Mohamed AJ: Analysis of the Outcome of Total Knee Replacement in Obese and Overweight Patients with Varus and Valgus Deformities. Int. J. Sci. Stud. 2017; 5(5): 239–241.\n\nWorld Health Organization: Obesity: preventing and managing the global epidemic; WHO Technical Report Series.2000; 1–252.\n\nDeMik DE, Carender CN, Glass NA, et al.: Are surgeons still performing primary total knee arthroplasty in the morbidly obese? Bone Joint J. 2021 Jun; 103-B(6 Supple A): 38–44. PubMed Abstract | Publisher Full Text\n\nMcElroy MJ, Pivec R, Issa K, et al.: The effects of obesity and morbid obesity on outcomes in TKA. J. Knee Surg. 2013; 26(2): 083–088. Publisher Full Text\n\nStickles B, Phillips L, Brox WT, et al.: Defining the relationship between obesity and total joint arthroplasty. Obes. Res. 2001; 9: 219–223. PubMed Abstract\n\nAndrew JG, Palan J, Kurup HV, et al.: Obesity in total hip replacement. J. Bone Joint Surg. Br. 2008; 90: 424–429.\n\nSantaguida PL, Hawker GA, Hudak PL, et al.: Patient characteristics affecting the prognosis of total hip and knee joint arthroplasty: a systematic review. Can. J. Surg. 2008; 51: 428–436.\n\nGanesan E, Balaji AB: Analysis of the Outcome of Total Knee Replacement in Obese and Overweight Patients with Varus and Valgus Deformities. Int. Res. J. Cli. Med. 2016; 1(3): 5–7.\n\nSuthar D, Vegad T: Assessment of total knee replacement in obese patients and non obese patients: a comparative study. Int. J. Res. Orthop. 2017; 3: 787–790. Publisher Full Text\n\nRodriguez-Merchan EC: The Influence of Obesity on the Outcome of TKR: Can the Impact of Obesity be justified from the Viewpoint of the Overall Health Care System? HSS J. 2014; 10(2): 167–170. PubMed Abstract | Publisher Full Text\n\nAbbas Z, Hafeez S, Naseem A, et al.: Effect of body mass index on duration of total knee replacement surgery: A prospective cross sectional study. Ann. Med. Surg. 2022; 82: 104637. PubMed Abstract | Publisher Full Text\n\nYasunaga H, Tsuchiya K, Matsuyama Y, et al.: Analysis of factors affecting operating time, postoperative complications, and length of stay for total knee arthroplasty: nationwide web-based survey. J. Orthop. Sci. 2009; 14: 10–16. PubMed Abstract | Publisher Full Text\n\nRavi B, Jenkinson R, O’Heireamhoin S, et al.: Leroux TS et al Surgical duration is associated with an increased risk of periprosthetic infection following total knee arthroplasty: A population-based retrospective cohort study. E Clin. Med. 2019; 16: 74–80.\n\nAl-Raddadi R, Bahijri SM, Jambi HA, et al.: The prevalence of obesity and overweight, associated demographic and lifestyle factors, and health status in the adult population of Jeddah, Saudi Arabia. Ther. Adv. Chronic Dis. 2019; 10: 204062231987899–204062231987810. Publisher Full Text\n\nAnandacoomarasamy A, Fransen M, March L: Obesity and the musculoskeletal system. Curr. Opin. Rheumatol. 2009; 21: 71–77. Publisher Full Text\n\nAlOmran A: The effect of Obesity on the Outcome of Total Knee Replacement (TKR). figshare. Journal Contribution. 2022. Publisher Full Text"
}
|
[
{
"id": "156959",
"date": "23 Dec 2022",
"name": "Dhanasekararaja Palanisami",
"expertise": [
"Reviewer Expertise Joint replacement 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\nObesity increases the risk of complications after TKR.\nThe two most common complications include a. Infection b. Aseptic loosening\nThere is no documentation of comorbid factors which could give a better idea about the patients who developed infection. eg. Diabetic status, Hb A1c level, serum albumin, smoking etc\nThere is no mention about the methods used for DVT prophylaxis. This can also influence the infection rate.\nThere are no details about incidence of tibial loosening or combined femoral and tibial loosening.\nThere are no details about the use of tibial stem, BMI cutoff for use of tibial stem.\nWe can safely say that obese patients are more prone for complications. But whether the authors followed the standard procedures to mimimise these complications is not evident in the data.\nIn conclusion the author advises to counsel the patients to reduce weight. What is the literature support to this claim? Is he advising bariatric surgery?\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": [
{
"c_id": "9166",
"date": "04 Jan 2023",
"name": "Abdullah AlOmran",
"role": "Author Response",
"response": "There is no documentation of comorbid factors which could give a better idea about the patients who developed infection. eg. Diabetic status, Hb A1c level, serum albumin, smoking etc Only two/10 patients with infection had type 2 diabetes mellitus which was controlled with HbA1c of 6.3. There is no mention about the methods used for DVT prophylaxis. This can also influence the infection rate. All patients received Enoxaparin sodium 40 mg subcutaneously daily for 14 days. There are no details about incidence of tibial loosening or combined femoral and tibial loosening. There are no details about the use of tibial stem, BMI cutoff for use of tibial stem. Tibial and femoral stem was used only in revisions of 75mm long. We can safely say that obese patients are more prone for complications. But whether the authors followed the standard procedures to mimimise these complications is not evident in the data. In conclusion the author advises to counsel the patients to reduce weight. What is the literature support to this claim? Is he advising bariatric surgery? By non-surgical methods."
}
]
}
] | 1
|
https://f1000research.com/articles/11-1406
|
https://f1000research.com/articles/11-1400/v1
|
29 Nov 22
|
{
"type": "Policy Brief",
"title": "Developing dispute resolution policy for conflicts settlement in public-private partnership (PPP) projects in educational institutions",
"authors": [
"Stephen Oyeyoade"
],
"abstract": "Background: United Nations Educational, Scientific and Cultural Organization (UNESCO) advocates a 15-20% allocation of the developing nations’ annual budget to educational development, but the available resources are not adequate to support the sector in the global south. Thus, University Hostel Development and Management (UHDM) policy was selected to attract private investors for the provision of educational facilities in Nigerian public universities. The policy considered a public-private partnership (PPP) model to ameliorate the menace of dilapidated infrastructures. Initially, there was massive investors’ participation until contractual disputes started to undermine the success recorded. Therefore, incessant disputes without resourceful resolutions motivate the need to examine this policy and spot the gaps for necessary improvement. Policy: The National Universities Commission (NUC) augments the provision of infrastructure in Nigerian public universities through the UHDM initiative in 2004. Conflicts owing to the multiplicity of stakeholders’ contrasting interests overwhelmed the initiative. Many value-added projects were terminated. The poor condition of facilities lingers to serve as a basic factor for half-baked graduates, low employee productivity and declined socio-economic values of the nation. This brief focuses on the role of formal dispute resolution mechanisms (DRMs) for conflict management in PPP arrangements. By observation, stakeholders rarely envisage potential conflicts, thus, no contemplation of specific DRMs adoption despite the inevitability of contractual disputes. Recommendations: This brief recommends the inclusion of the dispute resolution policy in the PPP Memorandum of Understanding (MoU). Recommendations are hereby generated from the analysis of the stakeholders’ opinions on the causes of the dispute, conflict prevention strategies, and the relevant dispute resolution mechanisms peculiar to the PPP contracts in educational institutions. Also, ambiguities in the policy that bordered on an inexplicit institutional framework, lack of PPP experts’ involvement, imperfect contract agreement, and deficiency of feasibility study, are resolved by the recommendations.",
"keywords": [
"Public-Private Partnership (PPP)",
"Contractual Disputes",
"Dispute Resolution Policy",
"Educational Institutions",
"Conflict Settlement Mechanisms"
],
"content": "Introduction\n\nThe failure of many developing countries to meet up with the UNESCO pronouncement on financing education in the global south cannot be overemphasized. Figure 1 indicates how the Nigerian annual budgets for education between 2015 and 2022 negate the UNESCO pronouncement due to the paucity of public resources.1 To salvage this invaluable sector, the National Universities Commission (NUC) instigated the University Hostel Development and Management (UHDM) initiative as a policy for flexible procurement plan for the delivery of educational facilities in 2004.2–4\n\nThis figure is reproduced with permission from Vanguard Newspaper Online - ASUU Strike: FG’s budgetary allocation to education lowest in 2022 - Report. https://www.vanguardngr.com/2022/08/asuu-strike-fgs-budgetary-allocation-to-education-lowest-in-2022-report/.\n\nThe adoption of public-private partnership (PPP) contracts for public universities occurs when the deterioration of the academic facilities was at the climax without a feasible remedy from the federal government. Not only that the existing facilities are insufficient but also not habitable. As of 2004, the aggregate of ‘on campus’ student hostels available in public universities can only accommodate less than 650,000 out of 1,780,667 students in school. So, the number of students residing off-campus outweighs those that are on campus.\n\nAccordingly, NUC’s adoption of PPP as a developmental tool gives precedence to the provision of student housing facilities hence the tag ‘UHDM’ for the initiative. The initiative was to moderate the wide deficit of facilities by encouraging private investors’ participation. Some universities embraced this private finance initiative (PFI) through PPP at an initial worth of US$ 240 million.5 Build-operate-transfer (BOT) as a model of PPP was adopted because of its consistent attributes for project construction, financing capacity for infrastructure development, and transferring of facilities in operational conditions at no cost to the procuring authority at the end of the concession term.6,7 These characteristics of BOT among others indicate its suitability for the objective of this scheme.\n\nHowever, educational infrastructure is known as a social good required to engender social benefits such as educational advancement that is meant to propel economic development in the long term. But private investors prefer investing in economically viable public goods that will produce a stream of income flows in no time.8 Given the divergence between the private investors’ short-term plan and the procuring authority’s long-term target, UHDM guidelines considered the flexibility of procurement terms and conditions as an important process to stimulate substantial participation of the private investors. This is also seen as a strategy for reducing the impact of risk factors and as well restricting the inherent conflicts in BOT operations among the stakeholders.\n\nPresently, conflicts have strained the relationship between private investors of PPP hostels and the procurement authorities in some universities.9 The lingering conflicts among the stakeholders have been putting several projects on hold or terminating them at the climax of the disputes. In 2014, Obafemi Awolowo University (OAU), Ile-Ife revoked the PPP agreements signed with 10 private investors due to conflicts bordering on the inability to complete the project within the regulated time, noncompliance with the agreed designs and specifications, and readjustment of the Memorandum of Understanding (MoU) in favour of the university management after many years of engagement, etc.\n\nIn a healthy business environment, the goal of every stakeholder is to ensure dispute resolution that will initiate decisions capable of ensuring project completion in a viable and sustainable manner while maintaining value for money.10 However, this objective had not been achievable based on the lack of adoption of a specific dispute resolution policy that will instil conflict settlement and forestall the awkward outcomes of failed contractual agreements.\n\nPrivate investors will feel encouraged to participate in PPP projects only when there is confidence in resolving disputes fairly and efficiently.11 It has been observed that an unresolvable dispute was one of the barriers to the investors’ participation.12 In other words, severe conflict defeats any PPP arrangement’s objectives. This factor thus points to the decline in private investors’ involvement in the PPP hostel concession. The aftereffect of this is the recurrence of the dearth of “on-campus” student accommodation that the implementation of UHDM initially solved.\n\n\nPolicy outcomes and implications\n\nThis brief is motivated by the need to have an enhanced knowledge of dispute resolution practices for effective management of influencing conflicts on the delivery of PPP contracts in educational institutions. Thus, the causes of disputes, conflict prevention strategies, and dispute resolution mechanisms are put into perspective. Also, the inclusion of a dispute resolution policy as an integral part of the PPP contract MoU guiding the roles of private investors and the procurement authority (individual university management) is thereby advocated.\n\nGlobally, this policy brief targets private investors/developers and procurement authorities (government ministries, departments, and agencies). More importantly, the private investors participating in PPP (build-operate-transfer - BOT) student hostel concessions in public universities, the universities’ managements (procurement authorities) adopting NUC policy on PPP models for the educational infrastructure development in Nigeria, and the NUC (the custodian of PPP policy) are the intended beneficiaries.\n\nAs laudable as the policy initiated by NUC on the adoption of the PPP model for the provision of educational infrastructure is, some unimaginable factors which curtailed the efficient delivery of concessional projects and the growth of the scheme. These factors constitute foundational challenges that evolve causes of PPP contractual disputes. Many researchers opined that conflict or disagreement is expectable in PPP contracts because of the multiplicity of stakeholders with diverse perceptions, long concession period, suspicion or distrust, and the prevalence of uncertainty and risk factors, among others.13–15 However, the rudimentary factors which are not considered at the formative stage of the policy subsequently placed the initiative on the difficult terrain that institute peculiar disputes among the stakeholders. Considering the five-point Likert scale responses of the sampled population of the PPP project participants as regards the efficacy of the policy on dispute resolutions, it observed that the shortcomings are connected to the following oversights occurred at the outset:\n\n• Lack of institutional framework\n\nNUC fails to institute clear guidelines for project tendering, evaluation, and award subject to the uniqueness of educational institutions. The individual university was left to the discretion of the procurement process resulting in imprecise procedures and, unequal terms and conditions. The existing guidelines of the Infrastructure Concession Regulatory Commission (ICRC)16 for PPP procurement have their limitations and are unable to address issues in PPP contracts of educational institutions.\n\n• Lack of expertise involvement\n\nAt the inception of policy development for UHDM, NUC fails to seek expert opinions that will facilitate an all-inclusive operational template for the adoption of the PPP model upon the peculiarity of the educational institutions.\n\n• Inexperience of the stakeholders\n\nIt was observed that many of the private investors as well as the procurement authorities (university management) lack cognate experience in the operational procedures of the PPP model. This condition fragmented the focus of the stakeholders.\n\n• Poor documentation\n\nDocumentation of PPP procurement processes and other proceedings are not considered. The importance of proper documentation is to serve as a reference and to allay controversies that may result from a misconception of ideas and conflicting interests of the stakeholders.\n\n• Over expectation\n\nThe NUC initiative and policy for adopting PPP raised the expectations of both the private investors and the procurement authorities (university management) beyond the attainable level. The expectations of the stakeholders on the socio-economic benefits continue to skew downwards due to unrealistic targets instigated by the policy.\n\n• Lack of modality for risk allocation\n\nNUC policy on PPP adoption is silent on the modality and the strength of equitable risk allocation. Comprehensive risk assessment of PPP contracts there is essential to unbiased risk sharing among the stakeholders without a template. Thus, inequitable risk allotment provokes disputes in PPP contracts.17\n\n• Indirection of MoU on conflict settlement\n\nThe content of the PPP contractual agreement or MoU must be all-inclusive. No aspect of the operational theme must be left out. Contrary to this opinion, the NUC policy on PPP failed to capture procedures for conflict settlements that can hinge on specific DRMs among other lacunae. In other words, the adopted MoU is clumsily devised by excluding strategies for dispute resolution. The unavailability of conflict resolution procedures is an alarming factor generating debriefing/termination of contract briefs, thereby clogging private investors’ participation and successful delivery of PPP projects in public universities.\n\n\nActionable recommendations\n\nVarious researchers of PPP projects asserted that conflicts are inevitable among the stakeholders. Some disputes are characteristically functional while others are dysfunctional.18 Those that are dysfunctional engender impaired relationships and eventual contract termination. The following findings suggest three areas where intervention can be achieved.\n\n• Identification of causes of dispute\n\nIn anticipation of operational intervention, understanding the root cause of the contractual dispute is crucial.19,20 Based on the Pareto principle,21 the author discovered that 20% of the factors instigating disputes in PPP contracts in public universities are significant while 80% of the causes are negligible. The result in Table 1 was elucidated based on the responses of the participants to five-point Likert scale questions, and the analysis done to determine the mean values of the variables.22 The table reveals the fundamental causes of dispute that necessitate earnest responsiveness of the NUC policy to developing a framework for preventing or settling PPP contractual conflicts.\n\nThe following causes of dispute are coded as CD19, CD3, CD 22, CD20, CD13, CD14, CD1, CD11, CD2, CD4, CD27, CD23, CD8, CD6, CD26, CD10, CD12, CD25, CD5, CD24, and CD7 are more differentiated among the 27 bases of conflict as indicated in Figure 2. The NUC policy ought to develop an underlying set of ideas to draw inferences from these chronological causes of dispute. This process will inform the utilization of workable dispute prevention strategies as well as the adoption of DRMs that are perceptive of the peculiarity of PPP contractual disputes.\n\n• Conflict prevention strategies\n\nThe advantages of preventing PPP contractual disputes are numerous. The cost of unprevented dysfunctional conflicts over the project delivery will leave stakeholders with immeasurable losses. It is rational to avert conflict than to pursue a resolution mechanism given that might not be cost-effective. The dispute resolution process will involve the consumption of resources (time, money, and energy) while the dispute prevention strategy will neutralize potential causes without snowballing.\n\nConflict prevention strategies are not contemplated as essential tools for avoiding disputes among the participants of PPP contracts by the NUC policy. However, the perspectives of 96 respondents on 13 dispute prevention strategies listed in Table 2, and the description of Figure 3 were rated on a five-point Likert scale. The relative importance index (RII) was used to rank the variables. The result suggests a sequential movement of engaging the prevention methods. This serves as a pointer for NUC policy amendment.\n\n• Dispute resolution mechanisms (DRMs)\n\nThe dispute resolution mechanisms materialize where dispute prevention strategies are unproductive.23 The choice of DRMs in settling dysfunctional disputes pivots on the analysis and the determination of three elements namely: familiarity (in terms of stakeholders’ awareness), frequency (in terms of DRMs constancy), and effectiveness (in terms of DRMs’ problem-solving capability). Figure 4 encapsulates stakeholders’ perceptions of seven DRMs accessible for dispute resolution in PPP contracts. In this regard, the NUC policy will be proactive by giving priority to mediation/reconciliation while deploying DRMs for PPP contractual dispute settlement. However, the policymaker must be conscious of the peculiarities associated with PPP concessions in educational institutions.\n\nSource: Author’s survey, unpublished 2022.\n\nSource: Author’s Survey, 2022.\n\nSource: Author’s Survey, 2022.\n\nDRMs have interrelated procedures that can be deployed separately or jointly utilized in settling conflicts.24 The functionality of DRM centres on specific paradigms that NUC policy ought to establish and understand by the participants. These models are as follows:\n\n• Values and beliefs\n\nThe dispute systems experts have argued that effective conflict management systems should be expansive, but priority should be placed on collaborative problem-solving, with ‘rights-based’ processes only brought into play as a last resort.25–27 Therefore, for any conflict management system to be productive, the respective rights and interests of all parties must be given priority. In other words, the stakeholders should be engaged in consensus-based processes that are responsive to various concerns as it affects individual values and beliefs.\n\n• Structure and roles\n\nEstablishing pertinent administrative and decision-making principles will strengthen DRMs in achieving the desired result.15,24 Among the guidelines or roles necessary to make DRMs workable are; swift responses to settling conflicts as they occur, adoption of multi-layered series of measures to resolve disputes appropriately, promoting the continuity of the stakeholders’ cordial relationship to ensure the steady running of the project, transparency, and accessibility must not be lagging in the implementation of DRMs for dispute settlements, and all the required resources such as manpower, time and money should be readily available to make DRMs function effectively.28 However, by institutionalizing these principles, the distinctive nature of the PPP project should be taken into cognizance as it affects educational institutions.\n\n• Procedures\n\nThe resourcefulness of DRMs lies in the range of approaches and procedures designed as the strategic inputs to ensure resolving the issues bother on the stakeholders’ differences. It is important to ascertain that justice, fairness, predetermination, preannouncement, consistency, and transparency take precedence in setting up DRMs procedures to arrive at all-inclusive decisions or resolutions acceptable to the conflicting parties.27\n\n• Skills and behaviours\n\nThe problem-solving skills of the DRM facilitator and attitudinal dispositions of the conflicting parties to the choice of DRM in settling the dispute will determine the success of the mechanism adopted. Understanding the contents of the dispute by the problem solver will pinpoint the suitable DRM and help obtain a satisfactory settlement for all the participants.24 Problem-solving techniques also required a dispute settler to possess strong behavioural and social skills that encompass good communication and persuasion skills. The possession of these skills will add an informal dimension that allows prompt resolution of disputes with little or no dependence on formal procedures.\n\nDeveloping DRMs that serve the interests of private developers and the university authorities engaging in PPP contracts is more demanding but imparting the foregoing paradigms into the DRMs structures will ensure the successful delivery of the educational infrastructure in the institutions of learning.\n\nVarious studies assert that disputes are inevitable in PPP contracts due to the dichotomy of the stakeholders’ interests among other factors. The present policy which ushered in PPP arrangements in public universities has no apparent aspect of conflict resolution that can guide the participating parties. However, if NUC policy will curtail dysfunctional PPP disputes, it must be capacitated via a far-reaching review as herein recommended:\n\n• Explicit institutional framework\n\nThe practicability of PPP concessions depends on a robust institutional framework.29 As it has been observed that the NUC policy lacks operational structure, it is, therefore, necessary to expedite clear guidelines for tendering processes, prequalification exercises, and standardized contract awards. A PPP administrative unit (PPP-AU) that will be specifically saddled with the PPP contract procurements should be established at each public university engaging PPP concessions. The unit will have adequate representation of all parties. The responsibility of the unit shall include the monitoring of every activity connected to service delivery and stakeholders’ contract relationships. In this regard, all potential dysfunctional disputes can be put in the spotlight, and the appropriate prevention measure can be taken, or where seems to be a possible escalation, a proactive DRM will be implemented to deter the degeneration of contract performance.\n\n• Involvement of PPP expertise\n\nIn every undertaking, expert opinions are essential where novices are challenged by the grey areas.30 Regardless of the far dated start-up period of the NUC policy on PPP concessions adopted by public universities, it is certain that the initiative (UHDM) is yet in its formative years given the absence of definite DRMs. This scenario is also responsible for the inability to surmount challenges accrued to contract disputes. Therefore, the policy must amplify the constitution of a think-tank at the centre. The think tank will serve as a central advisory board with its membership drawn from all categories of professionals in the construction industry and legal practice. This board will liaise with the PPP-AU of each institution from time to time as oversight functions. Any contract dispute rebutting PPP-AU interventions will be successfully managed by the central advisory board.\n\n• All-inclusive Memorandum of Understanding (MoU)\n\nThe importance of contract agreement or MoU in PPP concessions cannot be overemphasized. The articles or clauses guiding the rights and responsibilities of each participant are asserted in the MoU. Similarly, the scopes and or limitations of the concessions are equally affirmed. Since the NUC policy is silent on having a holistic template of MoU, each university resorted to a unilateral PPP agreement which is myopia about dispute resolution among others. Therefore, solving this shortcoming is long overdue. It is an urgent responsibility of the NUC to develop a unified dispute-resolution policy that will be integral to the MoU intended for the regulation of PPP contracts in educational institutions.\n\n• Mandatory PPP project feasibility study\n\nThe policy should emphasize project evaluation as a prerequisite to PPP contract award. The over-expectation of private investors is influenced by the quest for profit maximization. On the other hand, the procurement authorities will want to forestall any cutthroat profitability that can negatively affect the interests of the end-users of the PPP infrastructure. To prevent disputes related to the stakeholders over expectations, the execution of feasibility studies must be compelled on the parties through the policy.31 Thus, the feasibility study report will set the limit for the expectations of individual participants, and the potential disputes in this regard will be avoided.\n\nWhere common resources are insufficient and there is a need for public accountability on the provision of social infrastructures, PPP becomes a means of delivering governance dividends to the public the world over. This report about developing a dispute resolution policy is an idea that could be generalized and assist the global south countries engaging in various PPP models for infrastructure development. Many moribund PPP projects in developing countries are due to disputes caused by political interference, unrealistic concession periods, and volatile economy among others.32\n\nPPP contractual disputes will continue to emerge if the interests of the stakeholders remain varied. Thus, developing relevant policies that will prevent or mediate disputes should be given attention in the PPP agreements.\n\nConsidering the uniqueness of the DRMs, individual PPP procurement authorities and private investors can contrive and domesticate dispute resolution frameworks to conform to the extant regulations of the subject domain. Where resolution mechanisms are available, it is not outrageous to revamp the existing structure in line with the peculiarity of any given PPP project that is more of social values than economic values. PPP adopted for the educational sector requires workable interventions on dispute settlements because of its social characteristics that may not attract private investors who would be predisposed to economic returns rather than social services.\n\n\nConclusion\n\nIn the instance of the paucity of public resources to finance the education sector in Nigeria, it is commendable that NUC devised an initiative known as UHDM, to mitigate the shortage of facilities restricting the good quality of academic performance in public universities. However, the imperfection of the policy on which the adoption of PPP was founded undermined the gains of the initiative over the years. The emergence of peculiar contract disputes among the stakeholders without a clean and clear line of getting solutions via viable dispute resolution policy has instigated a decline in project delivery and private investors’ participation. To proffer lasting remedies, this policy brief has examined the causes of disputes and possible dispute prevention strategies. Likewise, the perception of the stakeholders on the utilization of DRMs has pinpointed how dispute resolution policy can be developed.\n\nConsequently, NUC is admonished to hastily embark on the overhauling of its PPP policy in line with the recommendations of this policy brief. It is also imperative to sensitize the universities management (procurement authorities) and other stakeholders on the policy implementation. However, the success of the PPP concessions adopted by the NUC for educational institutions is a function of an in-depth policy, and this must be attained to achieve the objectives of the UHDM initiative.",
"appendix": "Data availability\n\nFigshare: PPP Dispute Resolution Mechanism.sav. https://doi.org/10.6084/m9.figshare.21517284.v2. 33\n\nThis project contains the following underlying data:\n\n• PPP Dispute Resolution Mechanism.sav (the file includes data on i.) causes of dispute in PPP; ii.) dispute prevention strategies; and dispute resolution mechanisms in PPP).\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\nVanguard Newspapers Online: ASUU Strike: FG’s budgetary allocation to education lowest in 2022 - Report.2022. Accessed on 09/08/2022.Reference Source\n\nOkebukola P, Abdullahi I, Balogun B, et al.: Private Sector Participation in University Hostel Development and Management. Nigerian Universities Commission (NUC), Monograph Series. 2004; 1(4): 1–30.\n\nOkebukola P: Principles and policies Guiding current Reforms in Nigerian Universities. JHEA/RESA. 2006; 4(1); 25–36. 0851-7762. council for the Development of Social Science Research in Africa.\n\nBamiro OA: Sustainable Financing of Higher Education in Nigeria: Funding Models. A paper presented at the two-day consultative policy Dialogue by the committee of vice-chancellors (CVC) and Trust Africa, a Dakar-Senegal based H. E Development partner on the theme: The Future and relevance of Nigeria Universities and other Tertiary Institutions: Toward Higher Education Transformation, November 6-7. 2012.\n\nGbadegesin JT, Aluko BT: PPP/PFI for Financing Infrastructure in Public Tertiary Institutions in Nigeria. Journal of Built Environment Projects and Asset Management. 2014; 4(2).\n\nYuan J, Skibniewski MJ, Li Q: Managing the Performance of Public-Private Partnership Projects to Achieve Value for Money: Key Performance Indicators Selection. International Conference on Multi-National Construction Projects (“Securing High Performance Through Cultural Awareness and Dispute Avoidance”). Shanghai, China. November 21 – 23. 2008.\n\nBatra R: A Thematic Analysis to Identify Barriers, Gaps, and Challenges for the Implementation of Public-Private-Partnerships in Housing. Habitat International. 2021; 118: 102454. Publisher Full Text\n\nBitrina D, Diyamett K, Diyamett LDL:The Role of Public-Private Partnership in Innovation Development: Lesson from Africa. ORF Issue Brief No. 283. Observer Research Foundation;2019.\n\nOpawole A, Kajimo-Shakantu K: Assessment of Build-Operate-Transfer Model for Hostel Facilities Procurement in the Nigerian Public Universities. Proceedings of International Conference on Education, Development & Innovation. 2018.\n\nSanni GA, Adebiyi JO: Dispute Resolution in Public-Private Partnerships (PPPS) Contracts in Nigeria. Nigerian Research Journal of Engineering and Environmental Sciences. 2017; 2(1): 259–269.\n\nAbdul-Quium ASM: Legal framework for PPPs laws, contracts, and dispute resolution.2010. Accessed on 17/03/2022. Reference Source\n\nOpawole A, Kajimo-Shakantu K: Assessment of Build-Operate-Transfer Model for Hostel Facilities Procurement in the Nigerian Public Universities. Proceedings of International Conference on Education, Development & Innovation. 2018.\n\nGlobal Infrastructure Hub: Managing PPP Contracts After Financial Close: Practical guidance for governments managing PPP contracts, informed by real-life project data. Turner & Townsend;2018. Online Resource. 978-0-6480762-0-9.\n\nAhatty S, Ndekugri IE, Adaku E, et al.: Dispute Resolution in Public Private Partnership (PPP) Infrastructure Projects in Nigeria: Literature Review. International Journal of Engineering and Technology (IJERT). 2021; 10(09): September-2021. ISSN: 2278-0181.\n\nOsei-Kyei R, Chan APC: Conflict Management in Public-Private Partnership Projects. International Best Practices of Public-Private Partnership. 2021. ISBN 978-981-33-6267-3, ISBN 978-981-33-6268-0 (eBook). Publisher Full Text\n\nICRC Public Private Partnership Regulations: Infrastructure Concession Regulatory Commission (Establishment, etc) Act 2005 (Act No. 18 of 2005).2014.Reference Source\n\nBabatunde SO: Developing Public Private Partnership strategy for infrastructure Delivery in Nigeria. Northumbria University;2015. Doctoral Thesis.Reference Source\n\nIPPR: Call for evidence: Consultation paper on public-private partnerships.2000. Accessed on 17/03/2022.Reference Source\n\nDiaz Reus LLP: Resolving Disputes in Private/Public Partnership Agreements.2019. Accessed on 18/07/2022.Reference Source\n\nZheng X, Liu Y, Sun R, et al.: Understanding the Decisive Causes of PPP Project Disputes in China. Buildings. 2021; 11: 646. Publisher Full Text\n\nWaziri BS: Pareto Analysis of critical Risk factors of Build, Operate, and Transfer (BOT) projects in Nigeria. Journal of Construction Business and Management (JCBM). 2018; 2(1): 33–40. Publisher Full Text Reference Source\n\nOyeyoade SF:PPP Dispute Resolution Mechanism.sav. figshare. Dataset.2022. Publisher Full Text\n\nHarisankar KS, Screeparvathy G: Rethinking Dispute in Public-Private Partnerships for Infrastructure Development in India. Journal of Infrastructure Development. 2013; 5(1): 21–32. Publisher Full Text Reference Source\n\nCurrie D, Teague P: Conflict Management in Public-Private Partnerships: The Case of the London Underground. Negotiation Journal. 2015; 31: 237–266. Publisher Full Text\n\nCostantino C, Merchant C: Designing conflict management systems: A Guide to creating productive and healthy organizations. Thousand Oaks, CA:Jossey-Bass;1996.\n\nRowe M: Dispute resolution in the non-union environment. Frontiers in dispute resolution in labour relations and human resources. Gleason S, editor.East Lansing:Michigan State University Press;1997.\n\nSlaikeu K, Hasson R: Controlling the costs of conflict. San Francisco, CA:Jossey-Bass;1998.\n\nLynch J: CCRA: Contemporary conflict resolution approaches. The Revenue Canada Study of Innovations in Conflict Management. Ottawa:Canada Customs and Revenue;1998.\n\nUNESCAP: Promoting effective Public-Private Partnerships for Infrastructure Development. United Nations Economic and Social Commission for Asia and the Pacific. 2017. Policy Briefs No. 45, April 2017.\n\nBengesi KMK, Mwesiga P, Mrema T: Public Private Partnership in Tanzania’s Transportation Infrastructure: the way PPP is Understood, Challenges and the Way Forward. Economic and Social Research Foundation;2016. ESRF Policy Brief No. 2/2016.\n\nThe Hamilton Project: Public-Private Partnerships to Revamp U. S Infrastructure. The Brookings Institution;2011. Policy Brief 2011-02.\n\nSanni GA, Adebiyi JO: Dispute Resolution in Public-Private Partnerships (PPPS) Contracts in Nigeria. Nigerian Research Journal of Engineering and Environmental Sciences. 2017; 2(1): 259–269.\n\nOyeyoade SF:PPP Dispute Resolution Mechanism.sav. figshare. Dataset.2022. Publisher Full Text"
}
|
[
{
"id": "174797",
"date": "09 Jun 2023",
"name": "Kumar Neeraj Jha",
"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\nOverall review of paper: The problem statement given by paper is very much relevant to and require to be researched upon. Study design adopted by author is not satisfactory. Overall body of paper requires improvement.\nDetailed review:\nTitle: Title of the paper is clearly defined with reference to manuscript\n\nAbstract: Objectives are not clearly defined; author has not given any information on research methodology adopted in paper. Also, the abbreviations or terminology used like CD (Conflict Disputes), DRM (Dispute Resolution Mechanisms) and DPS (Dispute Prevention Strategies) are different in abstract and main paper. Paper has irrelevant information related to literature, however very less information related to research design. If abstract is read alone, it will not be able to Provide sufficient information about research. The use of words like \"are resolved by recommendations\" is not in purview of research. Keywords cited are also lacking key terms like UHDM, DPS.\n\nIntroduction: Introduction clearly describes the current situation and requirement as well as importance of the study to be carried out. However, the research questions and objectives are not defined. Citation of Figure 1 is not done properly. There are many texts in introduction and policy outcomes which need to be cited by the author. Many texts given in paper like \"Lack of institutional framework\", \"Lack of expertise development\" and \"Inexperience of stakeholders\" seems to be conclusive statements made by author without any reference or citations given.\n\nMethod and Results: Author has taken three different variables cause of disputes (Table 1), dispute prevention strategies (Table 2) and dispute resolution mechanisms (Figure 4). Author has not given any procedure of shortlisting these factors which were considered for study, nor has shown any relevance to PPP in education sector. Data collection and selection of method of analysis is not exhibited at all. There is no clarity on demography of sample population considered. The method adopted is not sufficient to achieve the problem statement of study. Further study and analysis of statistical methods is required. The author has not established any correlation between the cause of disputes with dispute prevention techniques and dispute resolution mechanisms which would have been more relevant to the research.\n\nDiscussions and Conclusion: The conclusion should have shown the relevance of DRM and DPS on conflict of disputes which are most likely to incur in the projects related to research. How to successfully implement DRM and DPS should also have been discussed?\n\nTables and figures: Tables and figures citing data analysis are not properly explained. Figure 2 is just graphical form of Table 1.\n\nReferences: Proper citations is not done in many places in paper.\nRecommendation: Major revisions are required for consideration of the paper.\n\nDoes the paper provide a comprehensive overview of the policy and the context of its implementation in a way which is accessible to a general reader? Partly\n\nIs the discussion on the implications clearly and accurately presented and does it cite the current literature? Partly\n\nAre the recommendations made clear, balanced, and justified on the basis of the presented arguments? No",
"responses": [
{
"c_id": "9757",
"date": "13 Jun 2023",
"name": "Stephen Oyeyoade",
"role": "Author Response",
"response": "The reviewer's comments are appreciated. The needful will be done to improve the article. Thank you"
}
]
},
{
"id": "174792",
"date": "17 Jul 2023",
"name": "Omid M. Rouhani",
"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 policy brief focuses on the role of formal dispute resolution mechanisms (DRMs) for conflict management in PPP arrangements, in the Nigerian public education context. The policy brief is relatively organized, it offers clear and useful recommendations for the case study, but comes short in several aspects, including, (i) a section/part explaining the case specific results is missing as well as current/proposed policies, (ii) figures/tables should be explained further, (iii) the survey needs to be discussed (who, when, how, …), and (iv) the lessons to developing economies section and the conclusion section are both very broad, in fact, no case-specific lesson is mentioned! and (v) the policy brief needs proof reading.\nMy major comments:\nFirst, the case specific results should be explained further. In the next point, I discuss what is missing regarding the representations of figures/tables. In addition to that, the author needs to explain the case-specific contexts/projects. Most of the strategies/mechanisms are very general. Several questions remained unanswered: What was different from other case studies? What are the examples of failures (case by case)? What are the current policies and what are their shortcomings?\n\nSecond, the author should elaborate on the observations from tables and figures. For instance, in Table 1, what is(are) the most important causes of dispute (Excessive contract sums variation), what are these, why they result in dispute, etc.\n\nThird, the author refers to a survey. S/he needs to discuss the survey (who are respondents, how are they chosen? When is the survey conducted?, how many responses, questions, …). Without such information, it is difficult to interpret the results.\nFourth, the lessons to developing economies section is very general, i.e., are any specific lessons mentioned? The discussions, like many other sections, do not elaborate on current and future policies. What are specific lessons from this study remains unanswered! Similarly, the conclusion section does not provide useful new information that explain the study.\n\nFifth, the paper needs proof reading as I mentioned in detail in the minor suggestions section below. Many parts/sentences are repetitive and long.\nMinor suggestions:\nMany parts require proof reading, in specific, several sentences are long, making the brief difficult to read. You need to break down sentences into two or more. For instance: “However, this objective had not been achievable based on the lack of adoption of a specific dispute resolution policy that will instil (instill) conflict settlement and forestall the awkward outcomes of failed contractual agreements.”\nOr “Considering the five-point Likert scale responses of the sampled population of the PPP project participants as regards the efficacy of the policy on dispute resolutions, it (is) observed that the shortcomings are connected to the following oversights occurred at the outset:” the author needs to break these sentences into two or more.\n\nThe author might want to add a section on current/future policies. Also, you might want to add a section describing the survey.\n\nSeveral relevant references are missing:\nGeneral PPP knowledge:\nYescombe, E.R., 2011. Public-private partnerships: principles of policy and finance. Elsevier.\nRegarding conflict resolution:\nOsei-Kyei, R., Chan, A.P., Yu, Y., Chen, C. and Dansoh, A., 2019. Root causes of conflict and conflict resolution mechanisms in public-private partnerships: Comparative study between Ghana and China. Cities, 87, pp.185-195.\nChou, J.S. and Lin, C., 2013. Predicting disputes in public-private partnership projects: Classification and ensemble models. Journal of Computing in Civil Engineering, 27(1), pp.51-60.\nRegarding risk sharing:\nDewatripont, M. and Legros, P., 2005. Public-private partnerships: contract design and risk transfer. EIB papers, 10(1), pp.120-145.\nRouhani, O.M., Geddes, R.R., Do, W., Gao, H.O. and Beheshtian, A., 2018. Revenue-risk-sharing approaches for public-private partnership provision of highway facilities. Case Studies on Transport Policy, 6(4), pp.439-448.\nRegarding institutional framework: Koch, C. and Buser, M., 2006. Emerging metagovernance as an institutional framework for public private partnership networks in Denmark. International Journal of Project Management, 24(7), pp.548-556.\n\nDoes the paper provide a comprehensive overview of the policy and the context of its implementation in a way which is accessible to a general reader? Partly\n\nIs the discussion on the implications clearly and accurately presented and does it cite the current literature? Partly\n\nAre the recommendations made clear, balanced, and justified on the basis of the presented arguments? Partly",
"responses": [
{
"c_id": "9921",
"date": "18 Jul 2023",
"name": "Stephen Oyeyoade",
"role": "Author Response",
"response": "The reviewer's comments are appreciated. The needful will be done to improve the article. Thank you"
}
]
}
] | 1
|
https://f1000research.com/articles/11-1400
|
https://f1000research.com/articles/11-728/v1
|
01 Jul 22
|
{
"type": "Research Article",
"title": "A hospital-based prospective cohort study to assess the factors associated with transmission dynamics of SARS CoV-2 among healthcare workers in Delhi",
"authors": [
"Mridu Dudeja",
"Pragya Sharma",
"Farzana Islam",
"Aqsa Shaikh",
"Farishta Hannah D. Singh",
"Yasir Alvi",
"Varun Kashyap",
"Warisha Mariam",
"Ayan Kumar Das",
"Safa Fazal Haque",
"Vishal Kumar Singh",
"Mohammad Ahmad",
"Anisur Rahman",
"Mridu Dudeja",
"Pragya Sharma",
"Farzana Islam",
"Aqsa Shaikh",
"Yasir Alvi",
"Varun Kashyap",
"Warisha Mariam",
"Ayan Kumar Das",
"Safa Fazal Haque",
"Vishal Kumar Singh",
"Mohammad Ahmad",
"Anisur Rahman"
],
"abstract": "Background: Healthcare personnel providing COVID-19 care are at increased risk of acquiring infection. Understanding the factors associated with the transmission of infection amongst healthcare workers provides input for the development of prevention strategies. The objectives of this study were to study the adherence to Infection Prevention and Control (IPC) measures followed between different categories of healthcare workers, to estimate seroconversion rate based on the type of exposure with COVID-19 patients and to study the association between seroconversion and IPC practices. Methods: A prospective cohort study was conducted from December 2020 to June 2021 among the healthcare workers in two tertiary healthcare institutes selected by purposive sampling. All Healthcare workers (HCW) participating in the clinical management or having any exposure to a laboratory-confirmed COVID-19 case were included. A total of 817 subjects were enrolled in the study. At baseline, details on IPC training and adherence along with details of the type of exposure with the COVID-19 patient were collected. The end-line visit was scheduled at 22-31 (preferably 28 days) days from the first visit for the collection of the symptom diary and end-line form. Results: Hand hygiene practices were found to be best among paramedics (98.0%), followed by doctors (84.5%) and nurses (90.1%). Maximum HCWs (99.5%; 99.8%; 97.1%) reported using Personal Protective Equipment (PPE) appropriately and regular availability of PPE in the hospital setting. Among the various categories of HCWs, nurses had the highest proportion (28.7%) of untrained personnel. Conclusion: The HCWs who had contact with the surroundings of an infected patient showed higher odds of seroconversion although not statistically significant. Further, analyzing the types of PPE used, we found that the use of masks and gloves were protective in preventing infection. Strengthening IPC training through refresher training and demonstrating the correct use of PPE can enhance adherence to IPC measures.",
"keywords": [
"COVID-19. Health-care workers",
"Infection Prevention and Control",
"seroconversion",
"transmission"
],
"content": "Introduction\n\nHealthcare workers are at the heart of a global crisis such as the COVID-19 pandemic. So far thousands of healthcare workers; including doctors and other healthcare workers have succumbed to COVID-19 in India. The virus traces back to 31st December 2019; when it was first discovered and later renamed as SARS-CoV-2 by the International Committee on Taxonomy of Viruses. At present, the strains prevalent are the alpha, beta, gamma, delta and omicron variants which are of notable importance.1 Coronaviruses transmits from one host to another host, either by an aerosol, fomite or via faecal-oral route.1 Globally, till January 2022, there have been nearly 5,614,909 confirmed deaths and more than 352,165,853 confirmed cases due to the pandemic.2\n\nIndia is second in line with the highest number of COVID-19 cases in the world with 39,543,328 cases and 489,896 deaths to date; just behind the United States of America and ahead of Brazil.3 The nucleus of the strategic amalgamation of all policies and stratagem, the national capital, stirs a discrete figure.4 The second wave, in April-May of 2021, saw an increase in the number of cases and deaths mostly in a phasedwise manner; weaning and waxing off after a sudden short and high peak.\n\nIf there is even the slightest breakdown in personal protection, health care providers providing COVID-19 care are at elevated risk of infection. Patients' clinical treatment and the execution of effective Infection Prevention and Control (IPC) procedures in health care facilities are both responsibilities of health personnel.5 Aside from the increased psychological strain caused by increased patient care and a lack of empathy, their overall well-being is a pressing concern. From line listing through diagnosis, treatment, rehabilitation, home visits, and prevention, the health workforce must be always on the front lines. Financial insecurity, violence, the rage of impacted families, and state mishandling all contribute to the misery.6\n\nSeveral advisories and directives have been updated by the Emergency Medical Relief Division of the Ministry of Health and Family Welfare, for managing Health care workers who are a valuable and scarce resource. They cannot be spared from being quarantined and treated in isolation. As a result, hospital mechanisms for activating the Hospital Infection Control Committee (HICC), as well as nodal officers assigned to respond to Health Associated Infections (HAIs), must strictly follow the guidelines as updated and made available on the Ministry of Health & Family Welfare (MoHFW) website.7,8\n\nDespite multiple studies in the field, there is considerable ambiguity among healthcare personnel due to a lack of concrete knowledge and its dissemination regarding the risk of transmission, the location of the mutation, vaccination efficacy, and political measures for COVID-19 and its variants.9 Understanding the factors associated with the transmission of infection amongst healthcare workers could provide valuable input for the development of suitable prevention strategies.\n\nHence the objectives of this study were:\n\n1. To study the adherence to IPC measures followed between different categories of healthcare workers.\n\n2. To estimate seroconversion rate based on the type of exposure with COVID-19 patients among healthcare workers.\n\n3. To study the association between seroconversion and IPC practices.\n\n\nMethods\n\nThis was a prospective cohort study conducted over seven months, from December 2020 to June 2021. This period covered India’s intense second wave of the COVID-19 pandemic.\n\nThe study was conducted among the health care workers of two tertiary health care institutes selected by purposive sampling. The first hospital was a government health facility, Lok Nayak Hospital in Central Delhi, which served as a dedicated COVID-19 tertiary care hospital for severe cases of COVID-19, with over 2000 beds and more than 1400 doctors and nurses providing care to COVID-19 patients. The second, Hakeem Abdul Hameed Centenary (HAHC) Hospital, is a trust hospital located in Southeast Delhi. It is a 200 bedded COVID-19 Care Hospital and has 1050 registered healthcare workers. The study population included all the health personnel who were working in this hospital and had come in contact or been exposed recently to a COVID-19 patient while providing them treatment and care.\n\nInclusion criteria\n\nAll HCW participating in the clinical management of a laboratory-confirmed COVID-19 case or having any of the following exposures with a COVID-19 case\n\n• Close contact (within 1 m) to a patient of COVID-19\n\n• exposed to infected patient's blood or body fluids,\n\n• exposed to infected patient's used materials, devices, or equipment,\n\n• contact with the environmental surface around the infected patient including his/her bed, table, wheelchair, ward corridor etc.\n\nThey were enrolled irrespective of their use of Personal Protection Equipment (PPE), any symptoms, or vaccination status.\n\nExclusion criteria\n\n• Those who had already suffered from COVID-19 before the start of the study\n\n• Any health worker who has a laboratory-confirmed COVID-19 case among their household/close contacts.\n\n• Clinically serious individuals who could not participate in the study.\n\nThis study provides information on the extent of COVID-19 infection among health workers and its risk factors. According to a study by Korth et al.10 in Germany, the estimated prevalence of SARS-CoV-2 in high- and low-risk HCWs was estimated to be 5.4% and 1.2%, respectively.9 The data reported by this study was used to calculate the sample size by OpenEpi Calculator Version 3.01. At 95% confidence level, 80% power, assuming a ratio of unexposed and exposed to be 1 (since exposure could not be determined in those using PPE) and expecting infection in the exposed group to be 5.4% and unexposed group to be 1.2%, the sample size was calculated to be 566 using the Fleiss formula. Furthermore, accounting for a 20% loss to follow-up, the effective sample size was 738. Thus, a total of 817 subjects were enrolled in the study.\n\nThe duty roster of all healthcare workers deployed in the COVID-19 wards of two tertiary care hospitals was extracted from the administrative office. Thereafter a random sample of HCWs was chosen from this list and assessed for eligibility. Those who fit the inclusion criteria were approached to provide written informed consent. At baseline, a questionnaire was administered by the researcher covering socio-demographic information, IPC training and adherence along with details of the type of exposure with the COVID-19 patient. The health care providers were also observed on their adherence to IPC protocols. With the launch of the vaccination drive in January 2021, details of vaccination were also enquired into. The completed questionnaire was entered in a Microsoft Excel sheet and checked by a supervisor against the hard copy for accuracy of data entry. The participants were provided with a symptom diary to note the development of any symptoms during the 21 days follow-up period. Regular follow up was done telephonically to ensure compliance. The end-line visit was scheduled at 22-31 (preferably 28 days) days from the first visit, for the collection of the symptom diary and end-line form. Serum samples were collected at baseline and at end-line to check for the presence of COVID-19 antibodies. The serum samples were tested for antibodies against COVID-19 using the WANTAI serological testing kit. It is a semi-quantitative kit and a value of above 1 was considered as positive.\n\n2-3 ml of blood was collected by venipuncture from all healthcare workers who were enrolled and provided written informed consent. The first sample was taken at enrollment, which was the baseline sample. If the sample tested negative for anti-SARS COV2 antibodies, a second sample was collected after 21 days. The paired samples testing protocol helped in the detection of asymptomatic carriers and understanding the pattern of seroconversion. Appropriate PPE was worn during sample collection and all the staff were well trained in safe specimen handling practices and spill decontamination protocols.\n\nThe sample collected appropriately was labelled and the blood vial was allowed to stand upright for 30 minutes at room temperature followed by centrifugation at 2500 rpm for five minutes. The samples were then sent to the testing laboratory after placing the vials in a carrier box in an upright position. Before accepting, the samples were checked for appropriate labelling, correct test request, sample adequacy, absence of leakage and haemolysis. The received samples were then centrifuged again and their serum separated in fresh labelled microcentrifuge tubes/cryo-vials and stored at -20°C before testing.\n\nWantai SARS-CoV-2-Ab ELISA kit (Lot no.: NCOA20200902) detects total antibodies against the SARS-CoV-2 virus and is based on the principle of two-step incubation antigen “sandwich” enzyme immunoassay. Briefly, 100 ml of the patient’s serum is added to polystyrene microwell strips pre-coated with recombinant SARS-CoV-2 antigen. Three wells are marked as negative calibrators and two wells as positive calibrators. 50 ml of negative and positive calibrator are added to respective wells and the plate is incubated at 37°C for 30 minutes. Post incubation the wells are washed five times with diluted wash buffer. 100 μl of HRP-Conjugate is then added to each well and the plate is incubated at 37°C for 30 minutes. The wells are washed again five times and 50 μl of Chromogen Solution A and then 50 μl of Chromogen Solution B is added into each well. The plate is then incubated at 37°C for 15 minutes in the dark. Each well receives 50 μl of Stop Solution, which is gently mixed. Absorbance is measured using PR4100 microplate reader, Bio-Rad, USA (dual filter) with reference wavelength at 600~650 nm. The cut-off value (C.O.) was calculated as C. O = Nc + 0.16 (Nc = the mean absorbance value for three negative calibrators). The tested serum samples were stored at -80°C with proper labelling.11\n\nNegative results were reported for specimens with absorbance (A) less than the Cut-off value, which meant that no SARS-CoV-2 antibodies were detected with WANTAI SARS-CoV-2 Ab ELISA (A/C.O. < 1). Specimens with absorbance equal to or greater than the cut-off value were considered positive, which indicated that SARS-CoV-2 antibodies were detected using WANTAI SARS-CoV-2 Ab ELISA A/C.O. ≥ 1). The reports were shared with the principal investigator electronically and all the study participants were provided with a copy of their results and were counselled accordingly.\n\nSeropositivity: For this study, we considered all the enrolled subjects who were positive (A/C.O. >1) for SARS-CoV-2 antibodies, detected with the WANTAI SARS-CoV-2 Ab ELISA kit at baseline. This included all those who were positive due to prior COVID-19 infection or because of vaccination against COVID-19.\n\nSeroconversion: For this study, we considered all the enrolled subjects who were negative (A/C.O. < 1) for SARS-CoV-2 antibodies, detected with the WANTAI SARS-CoV-2 Ab ELISA kit at baseline but subsequently tested positive in the end line serum sample collected between 22-31 (preferably 28 days).\n\nSecondary infection rate: Those HCW who were enrolled after a recent exposure with a COVID-19 patient AND confirmed for new infections of COVID-19, assessed through serological assays on paired samples, divided by susceptible contacts (total enrolled HCW). Due to the limitation of our study, it would be the same as the seroconversion rate.\n\nThese healthcare workers included medical staff like doctors, nurses, paramedical staff (which includes physiotherapists, occupational therapists and other allied health professionals, General Duty Assistants, housekeeping staff, security staff) students of medical, nursing and paramedical sciences and other front office staff who were directly involved in patient care.\n\nThe forms were verified for consistency and completeness. Collected data was entered and coded appropriately and later cleaned for any possible errors in Microsoft Excel. For analysis, SPSS (version 26) was used. The frequency tests were performed after determining clear values for the outcomes. Categorical data was presented as percentages (%). Descriptive analysis (time, place, and person) provided preliminary insight into the clinical spectrum and course of COVID-19 among health workers. Pearson’s chi-square and bivariate logistic regression were done to evaluate the independent associations of multiple factors. All tests were performed at a 5% level of significance, and hence a p-value of less than 0.05 (p-value < 0.05) was taken as a significant association.\n\nEthical considerations for doing the study were undertaken and all norms of confidentiality, autonomy, beneficence, and consent were taken care of. The study started after approval by the Research Project Advisory Committee (RPAC) and Institutional Ethics committee (IEC) of both Hamdard Institute of Medical Sciences and Research (HIMSR/IEC/004/2020/Dated: 01/09/2020) and Maulana Azad Medical College (F.1/IEC/MAMC/(79/07/2020/No. 203). Other necessary permissions from the hospital and medical colleges were obtained. Written informed consent in English/Hindi was obtained from each participant before their enrolment in the study.\n\nAll personnel involved in the study were trained in infection prevention and control procedures (standard, contact, droplet, and airborne precautions, as described by the World Health Organization (WHO) technical guidelines).8 Procedures included proper hand hygiene and the correct use of medical or respiratory face masks.\n\nConfidentiality was maintained by assigning a study identification number by the investigation team for the labelling of questionnaires and clinical specimens. The linking of this identification number to individuals was performed by the investigation team and will not be disclosed elsewhere. The data has been shared with the funding agency WHO, which also provided support for data analysis. The shared data only included the study identification number without any personally identifiable information.\n\n\nResults\n\nA total of 1210 HCWs were contacted of whom 817 were recruited in this study as per the inclusion criteria. All the participants were interviewed and blood sampling was done for serology at the baseline visit. Out of the 817, 384 were included at the end line serology assessment. The selection of participants and loss to follow up are highlighted in Figure 1.\n\nThe study had a roughly equal distribution of male and female participants, with a majority of them belonging to the 31-60 years age group. We had a good representation of doctors, nurses, and paramedics with the majority of them working in high-risk areas where the risk of transmission of COVID-19 was high such as COVID wards, Intensive Care Unit (ICU), testing areas etc. The COVID vaccination drive in India was launched during the study period in a phased manner, prioritizing the vaccination of HCW and frontline workers. Hence, about 50% of the participants were either partially or fully vaccinated. The participant characteristics have been shown in Table 1.\n\nTable 2 presents a summary of the results relating to IPC training and the attitudes towards IPC practices, as reported by various categories of HCWs. On comparing the training received for IPC, most healthcare workers, in all categories; doctors, nurses and paramedics, received less than 2 hours of IPC training and amongst them, nurses were seen to have the highest proportion. 26.8% of nurses had not received any IPC training in the past. Hand hygiene measures as recommended during their IPC training were practiced by the majority of HCWs of all categories, with paramedics (100) (98.0%) showing best adherence, followed by nurses (90.1%) and doctors (84.5 %). An interesting finding was that among the three categories of HCWs, almost all paramedics reported that they always followed hand hygiene as prescribed. However, doctors (84.5%) and nurses (90.1%) seemed to have fallen short in adhering to the prescribed hand hygiene measures at appropriate moments such as before and after touching a patient, their surroundings, or their body fluids. Maximum HCWs (99.5%; 99.8%; 97.1%) reported that they always wore their PPE appropriately and had a regular availability of PPE in the hospital setting.\n\nAt baseline, the serology of all HCWs was assessed, as shown in Figure 2. The presence of COVID-19 antibodies was either due to a previous infection or vaccination. The highest proportion was found amongst nurses followed by doctors and paramedics. After 22-31 days, at the end line serology, a positive seroconversion was observed in 53(14.1%) HCWs who were negative at the time of baseline sampling. Further, we analyzed the relation between seroconversion and vaccination status of the individuals and found them to be significantly associated with each other as shown in Figure 3. A positive seroconversion was noted most (29.8%) among the completely vaccinated group. Among those who were not vaccinated, seroconversion was observed amongst 9.9% of the participants.\n\nIn this study, we assessed the association of IPC attitudes amongst HCWs with their baseline serology status as seen in Table 3. For analysis, we classified the use of various IPC attitudes into dichotomous variables as adequate and inadequate. We found that among those that received IPC training, 45.5% (285) were negative and 54.5% (341) showed a positive result. Among those who reported that they wore PPE appropriately 45.9% (363) had a negative result and 54.1% (428) showed positive serology. None of the measures was found to have any significant association with serology, however, using appropriate PPE was shown to have a protective effect on preventing infection as seen by negative baseline serology but was not statistically significant.\n\nWe also assessed the association between IPC attitudes and seroconversion (Table 4), we found that those who had received training had a higher odd of seroconversion as compared to those who did not, this was however not statistically significant. It was seen that those who adhered to hand hygiene practices adequately had a lower odd of seroconversion although none of the measures was significant. Our study found that those who followed IPC standards had higher odds of seroconversion, in contrast to those who did not.\n\nFurther, as shown in Table 5, on assessing the seroconversion based on the type of exposure a healthcare worker had, we found that seroconversion was maximum among participants who had close contact (within one meter) of a COVID 19 patient (17.5%) or those who had a face-to-face exposure (34.4%). Those with contact with patient materials showed a statistically significant association with fewer patients showing seroconversion. However, HCWs who had contact with the surroundings of an infected patient seemed to show a higher odd of seroconversion even though it wasn’t found to be statistically significant. This could be due to the perception that infection cannot be acquired by contact with the surroundings and hence a laxity in the hand hygiene measures employed. Analyzing the kind of PPE used we found that the use of masks and gloves were protective in preventing infection. While the use of gowns/coveralls/headcover or shoe cover didn’t show any protective effect. The association of the type of contact and the kind of PPE used on seroconversion has been shown in Table 6. None of the measures was found to be statistically significant however the protection conferred by the use of masks, face shields and gloves were better in comparison to that of other PPE measures.\n\n\nDiscussion\n\nThis study was conducted to assess the Infection Prevention and Control measures amongst various categories of healthcare workers during the COVID-19 pandemic. To evaluate its effectiveness in preventing human-to-human transmission among recently exposed healthcare workers, we measured their seroconversion, which has been used as a proxy indicator for secondary infection in this study. We collected information about the exposure to COVID-19 infection and the IPC measures taken by them, along with the serological testing at baseline and after 3-4 weeks. This was further supported by a daily symptom diary. The launch of the vaccination campaign and the disastrous second wave of COVID-19 in New Delhi, coincided with the data collection period of this study. Therefore, the results need to be appreciated keeping in mind that approximately 50% of study participants received at least one dose of the vaccine. Due to vaccination, several individuals were positive at their baseline serology, hence as per the protocol, they had to be excluded (47.1%) at the beginning of the study. Further, we had a drop-out rate of 10.7%. Despite a considerable loss in the number of participants, the socio-demographic characteristics amongst the participants at baseline and end-line were very similar, thus making the groups relatively comparable.\n\nIn our study, we found a higher proportion of individuals of 31-60 years, as this age group comprises the working-age group. This distribution was also seen in other similar studies conducted amongst healthcare workers.12,13 Also, most of the HCWs above the age of 60 were asked to provide telehealth services to prevent them from getting infected, hence were not exposed to COVID-19 patients. We had a higher participation from non-physician healthcare workers, a majority being nurses. This could be due to either lack of time amongst physicians to participate in the study or, most of them having had previous COVID-19 infection or being vaccinated and hence excluded at baseline. We found that only 3/4th of the healthcare workers had been trained in Infection Prevention and Control measures. This could be due to new HCWs being recruited/hired to handle the surge in cases and not having had the opportunity to attend training. Tabah et al found a similar pattern with about 83% of their staff having adequate training, but desiring additional training for better compliance.13\n\nAn unusual finding was that the attitude towards IPC measures was found to be better amongst non-physicians such as nurses and paramedics as compared to doctors. This was despite doctors having received better training as compared to a quarter of the nursing staff not being trained in IPC measures. In contrast to our findings, a study among Bangladeshi HCWs found no difference in knowledge amongst physician and non-physician HCWs, however, they found, that physicians showed a better attitude towards IPC practices.14 A systematic review however uncovered a similar pattern, wherein the doctors showed poorer compliance as compared to nurses in following hand hygiene measures.15 Further, the attitude towards hand hygiene measures of the paramedical staff was seen to be the best, despite the finding that 1/4th of them did not know what the standard IPC precautions were. The WHO recommends 5 moments of hand hygiene to prevent the spread of hospital-acquired infections. However, in our study, we found that the hand hygiene measures showed a decreasing trend in compliance to the moments, depending on the risk of exposure and based on the nature of contact.16 The attitude towards ensuring hand hygiene was seen to be the highest after touching body fluids and was seen to be the least before touching a patient or after touching their surroundings. Although the adherence to wearing PPE was best amongst the paramedic staff, the availability amongst them seemed to be the lowest. Considering the rationale use of PPE in a scenario of shortage of PPEs during the surge of the pandemic, doctors and nurses were given greater priority over paramedics. Incorporating IPC training in the curricula of medical, nursing and paramedic students and conducting refresher training courses on IPC measures are essential to ensure better compliance, especially in settings such as a medical college where the staff are constantly changing.15,17 Adherence to IPC measures doesn’t happen naturally. Thus a multifaceted approach targeted to improve compliance, achieved through training, followed by observation and feedback is pivotal in improving adherence to IPC practices.15 Apart from training, building and promoting an IPC resilient system requires input from IPC professionals, thus building a career path in facilitating a behaviour change among HCWs at the state and national levels.17,18 Ensuring adequate supplies of PPE, sanitizers and handwashing stations, placed at easily visible and accessible places, could enhance the utilization of these resources.18\n\nThe chances of secondary infection were seen to be significantly higher among those with face-to-face exposure (p = 0.039), contact with patient body fluid fluids (p = 0.014), or patient materials (p < 0.001). This was surprising considering that these routes of transmission are less commonly recognized yet showed significant risk of infection. A laxity in ensuring hand hygiene practices and other IPC measures could have led to easy transmissibility. Further, the tendency of people to touch the infected mask or inappropriate doffing measures could be a plausible explanation.19 The more commonly known routes of transmission such as aerosolizing procedures or direct contact close to 1 meter of the patient, did not show any significant infections. Better awareness of these well-established routes of transmission could have warranted adequate IPC practices. Recent researches on identifying the routes of transmission for COVID-19 have disproved the airborne route of transmission and claimed a lower chance of transmission via the contact route.20,21 Thus, this could further explain the lower rates found in our study. Higher transmission from contact with surroundings could also imply poor sanitization of the patient surroundings. Hence, healthcare establishments should emphasize the importance of sanitizing surfaces as they act as fomites for transmission of several hospital-acquired infections.21,22 We cannot dismiss the possibility of contracting an infection from settings outside the workplace, such as the home or during lunch breaks. It is quite common for HCWs to take breaks together where they do not adhere to practices of physical distancing or wearing a mask.12 Thus ensuring reduction in other congregate settings and promoting hand hygiene and disinfection of surfaces in settings outside the workplace is also essential.23\n\nGood PPE practices and an alert mind, have been proven tools to prevent hospital-acquired infections.22 In our study, we had a similar finding, where appropriate use of masks and other elements of personal protection was seen to have a lower odd of secondary infection. Of all the types of PPE used, the use of masks was found to be most effective in decreasing transmission of infection. Similar results have been found in other studies where it was reported that the seropositivity among those wearing any type of mask (10.9%; 95% CI: 10.1%–11.6%) was significantly lower than for those without (17.5%; 95% CI: 16.0%–19.2%).24 Considering, the dearth of availability of PPE, promoting its rational use is crucial. Thus, while masks, gloves and hand hygiene practices are key components in any setting, other types of PPE such as shoe cover, head cover, goggles etc. can be reserved for high-risk areas. The mere availability of resources doesn’t guarantee its utilization, and if utilized then its appropriate utilization. Thus, demonstration of using PPE by trained infection control professionals could impress upon the HCWs the appropriate methods of usage and disposal.\n\nThis study was not without limitations. One of the biggest challenges we faced was the launch of the COVID-19 vaccination drive among healthcare workers. Although it was a much-awaited relief, it could have considerably confounded the findings of this study. It was not possible to differentiate between the seropositivity developed due to vaccination or due to secondary infection. The drawback of not using RT-PCR as a diagnostic tool for secondary infection was greatly felt. The use of RAT/RT-PCR could have enabled us in differentiating the true secondary infections from theseropositives which were due to COVID-19 vaccination. Finally, the IPC measures adopted were self-reported, hence the underlying fear of interviewer bias cannot be overlooked, especially amongst the paramedical staff who might have been apprehensive from fear of disciplinary action if the truth were told.\n\nIn conclusion, strengthening IPC practices is essential in and to the entire community. Healthcare workers being very much a part of the community need to ensure the appropriate use of personal protection to prevent contracting any disease and transmitting it to the community and vice versa. In view of the new knowledge added, rational and appropriate use of PPE and other disinfection practices need to be communicated to the larger audience. Further research on the transmission of COVID-19 in low- and middle-income settings is required.\n\n\nData availability\n\nThe data that support the findings of this study are subject to certain data-sharing restrictions. If any researcher is interested in finding out more about this study or wishes to collaborate with us, kindly send an email stating your intentions to the corresponding author. Due permission will be sought from WHO thereafter, and communicated likewise to the interested researchers.",
"appendix": "References\n\nAlphacoronavirus DN: Tidona C, Darai G, editors. The Springer Index of Viruses. New York, NY: Springer; 2011 [cited 2021 Jul 19]; p. 371–83. Publisher Full Text\n\nWorldometer: COVID-19 Coronavirus pandemic.[cited 2022 Jan 10]. Reference Source\n\nJohns Hopkins University: COVID-19 Dashboard by the Center for Systems Science and Engineering (CSSE) at Johns Hopkins University (JHU). Johns Hopkins Coronavirus Resource Center; [cited 2022 Jan 10]. Reference Source\n\nDudeja M, Shaikh A, Islam F, et al.: Assessment of Potential Risk Factors for COVID-19 among Health Care Workers in a Health Care Setting in Delhi, India - A Cohort Study. medRxiv. 2022 [cited 2022 Mar 16]; p. 2022.02.28.22271674. Publisher Full Text\n\nChou R, Dana T, Buckley DI, et al.: Epidemiology of and Risk Factors for Coronavirus Infection in Health Care Workers: A Living Rapid Review. Ann. Intern. Med. 2020 Jul 21; 173(2): 120–136. PubMed Abstract | Publisher Full Text\n\nSøvold LE, Naslund JA, Kousoulis AA, et al.: Prioritizing the Mental Health and Well-Being of Healthcare Workers: An Urgent Global Public Health Priority. Front. Public Health. 2021 [cited 2022 Mar 15]; 9. PubMed Abstract | Publisher Full Text\n\n33 doctors in over 4% Delhi health workers affected by coronavirus, situation ‘worrying’: Hindustan Times.2020 Apr 28 [cited 2022 Jan 10]. Reference Source\n\nGovernment of India Ministry of Health & Family Welfare: Revised Advisory for managing Health Care Workers (HCWs) working in COVID and Non-COVID areas of the Health Care Facilities.2022 Jan 9 [cited 2022 Jan 11]. Reference Source\n\nWorld Health Organization: Coronavirus disease (COVID-19) pandemic.[cited 2022 Jan 5]. Reference Source\n\nKorth J, Wilde B, Dolff S, et al.: SARS-CoV-2-specific antibody detection in healthcare workers in Germany with direct contact to COVID-19 patients. J. Clin. Virol. 2020 Jul; 128: 104437.\n\nWantai: COVID-19 Serology and Molecular Tests: Wantai BioPharm.[cited 2022 Jan 5]. Reference Source\n\nAl Maskari Z, Al Blushi A, Khamis F, et al.: Characteristics of healthcare workers infected with COVID-19: A cross-sectional observational study. Int. J. Infect. Dis. 2021 Jan; 102: 32–36. PubMed Abstract | Publisher Full Text\n\nTabah A, Ramanan M, Laupland KB, et al.: Personal protective equipment and intensive care unit healthcare worker safety in the COVID-19 era (PPE-SAFE): An international survey. J. Crit. Care. 2020 Oct; 59: 70–75. PubMed Abstract | Publisher Full Text\n\nHossain MA, Rashid MUB, Khan MAS, et al.: Healthcare Workers’ Knowledge, Attitude, and Practice Regarding Personal Protective Equipment for the Prevention of COVID-19. J. Multidiscip. Healthc. 2021; 14: 229–238. PubMed Abstract | Publisher Full Text\n\nAlhumaid S, Mutair AA, Alawi ZA, et al.: Knowledge of infection prevention and control among healthcare workers and factors influencing compliance: a systematic review. Antimicrob. Resist. Infect. Control. 2021 [cited 2022 Feb 19]; 10: 86. PubMed Abstract | Publisher Full Text Reference Source\n\nWorld Health Organization: WHO Guidelines on Hand Hygiene in Health Care: a Summary.2009. [cited 2022 Feb 8]. Reference Source\n\nPatel LN, Kozikott S, Ilboudo R, et al.: Safer primary healthcare facilities are needed to protect healthcare workers and maintain essential services: lessons learned from a multicountry COVID-19 emergency response initiative. BMJ. Glob. Health. 2021 [cited 2022 Feb 19]; 6(6). Publisher Full Text Reference Source\n\nTomczyk S, Storr J, Kilpatrick C, et al.: Infection prevention and control (IPC) implementation in low-resource settings: a qualitative analysis. Antimicrob. Resist. Infect. Control. 2021 [cited 2022 Feb 19]; 10: 113. PubMed Abstract | Publisher Full Text Reference Source\n\nGupta MK, Lipner SR: Personal protective equipment recommendations based on COVID-19 route of transmission. J. Am. Acad. Dermatol. 2020 Jul; 83(1): e45–e46. PubMed Abstract | Publisher Full Text\n\nJones RM: Relative contributions of transmission routes for COVID-19 among healthcare personnel providing patient care. J. Occup. Environ. Hyg. 2020 Sep; 17(9): 408–415.\n\nLai X, Wang M, Qin C, et al.: Coronavirus Disease 2019 (COVID-2019) Infection Among Health Care Workers and Implications for Prevention Measures in a Tertiary Hospital in Wuhan, China. JAMA Netw. Open. 2020 May 1; 3(5): e209666. PubMed Abstract | Publisher Full Text\n\nWong SCY, Kwong RT-S, Wu TC, et al.: Risk of nosocomial transmission of coronavirus disease 2019: an experience in a general ward setting in Hong Kong. J. Hosp. Infect. 2020 Jun; 105(2): 119–127. PubMed Abstract | Publisher Full Text\n\nKambhampati AK, O’Halloran AC, Whitaker M, et al.: COVID-19-Associated Hospitalizations Among Health Care Personnel - COVID-NET, 13 States, March 1-May 31, 2020. MMWR Morb. Mortal. Wkly Rep. 2020 Oct 30; 69(43): 1576–1583. PubMed Abstract | Publisher Full Text\n\nSims MD, Maine GN, Childers KL, et al.: Coronavirus Disease 2019 (COVID-19) Seropositivity and Asymptomatic Rates in Healthcare Workers Are Associated with Job Function and Masking. Clin. Infect. Dis. 2021 Jul 30; 73(Suppl 2): S154–S162. PubMed Abstract | Publisher Full Text"
}
|
[
{
"id": "143000",
"date": "19 Jul 2022",
"name": "Mohammad Shibly Khan",
"expertise": [
"Reviewer Expertise Primary health care",
"medical education",
"health systems 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\nThe authors have appropriately chosen the prospective study design, to assess the effectiveness of the IPC (Infection Prevention and Control) practices in controlling the transmission of SARS-CoV-2 among HCWs (healthcare workers). As the study was conducted in hospital setting, the possibility of other confounding factors could not be ruled out; which has been duly acknowledged as a limitation. However, it’s not been mentioned in the methodology whether there was any attempt to cross check the interplay of any such factors. The authors have documented self-reported IPC practices through researcher administered questionnaire and observing the IPC protocols, however, they have not specified how many participants were actually observed. Considering the possibility of social desirability bias, its pertinent to employ innovative data collection techniques apart from self-reported responses. For instance, Paul E. et. Al. have additionally video-recorded the observance of IPC protocols among their study participants.1 Nevertheless, the methodology and analysis have been meticulously presented, which allows the replicability.\n\nIs the work 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": "143001",
"date": "30 Sep 2022",
"name": "Mohd Maroof",
"expertise": [
"Reviewer Expertise epidemiology",
"geriatics"
],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 can't clearly give idea about what the study is about.\n\nIn abstract result section main findings are not provided.\n\nObjectives are not specific.\n\nMethodology section:\nWhat cohort included in study not clear, sample size came out to be 738, but why were 817 subjects enrolled?\n\nWhy simple random sampling method was used as many different types of HW (health workers) were enrolled (anything done to ensure appropriate representation of each HW type?)\n\n\"Descriptive analysis (time, place, and person) provided preliminary insight into the clinical spectrum and course of COVID-19 among health workers.\"- can't find these in results.\n\nCouldn't understand why All tables & figures \"n\" variable. table 5 \"Aerosolizing Procedure\" p- value & odds ratio doesn't correlate.\n\nOverall, results are not in line with objectives.\n\nConclusion and results doesn't correlate completely.\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": "8875",
"date": "29 Nov 2022",
"name": "farishta singh",
"role": "Author Response",
"response": "Respected Reviewer, Thanks for your valuable input and keen observations of the manuscript. We appreciate the time spent thoroughly reviewing the paper to improve the final manuscript. We have addressed your queries and incorporated your suggestions to the best of our abilities. Kindly find below the comments with our response. Reviewer Comment: \"The title can't clearly give an idea about what the study is about.\" Author's Response: Dear sir, the title of the study is ‘A hospital-based prospective cohort study to assess the factors associated with transmission dynamics of SARS CoV-2 among healthcare workers in Delhi’. We have tried to incorporate the study design, population, and outcome as much as possible, as it is essential for any title. We understand there might be some confusion about the term ‘transmission dynamics. However, due to the word limit in the title, all the parameters assessed to understand the transmission amongst healthcare workers have been summarized using the term ‘Transmission dynamics. Reviewer Comment: \"In abstract result section main findings are not provided.\" Author's Response: Thank you for the feedback, we have included the suggestions in the revised results section. We have taken your comment seriously and thoroughly revised the relevant section. Revised results in abstract: \"Hand hygiene practices were found to be best among paramedics (98.0%). Maximum HCWs (99.5%; 99.8%; 97.1%) reported using Personal Protective Equipment (PPE) appropriately and regular availability in the hospital. Seroconversion was maximum among participants who had close contact (within one meter) of a COVID-19 patient (17.5%) or those who had a face-to-face exposure (34.4%).\" Reviewer Comment: \"Objectives are not specific\" Author's Response: Respected sir, we have a different opinion on this. The objectives of the study are 1. To study the adherence to IPC measures followed between different categories of healthcare workers 2. To estimate seroconversion rate based on the type of exposure with COVID-19 patients among healthcare workers 3. To study the association between seroconversion and IPC practices. We find all the objectives specific and to some extent SMART. With regard to specificity, for the first objective, we looked at the IPC practices followed by the HCWs. For the second objective, we measured seroconversion across different HCWs and their type of exposures during recent contact with COVID-19 cases. And for the third objective, we assessed seroconversion and its independent factors including IPC practices. We are of the opinion that all the objectives are specific. Reviewer Comment: \"Methodology section: What cohort was included in the study is not clear, sample size came out to be 738, but why were 817 subjects enrolled?\" Author's Response: We are thankful for your feedback. The cohort included all the healthcare workers who were recently exposed to a COVID positive patient in the two tertiary care hospital setting where the study was conducted. We calculated the sample size as 738, but the study was open to enrollment and as several HCWs were exposed especially during the second wave, all were enrolled with the hope to have an adequate proportion of different categories of HCW, to increase the generalizability of the study. Reviewer Comment: \"Why simple random sampling method was used as many different types of HW (health workers) were enrolled (anything done to ensure appropriate representation of each HW type?)\" Author's Response: Thanks for pointing out this limitation. As this study was conducted during the COVID pandemic, due to the paucity of time, we felt simple random sampling would be more convenient as compared to stratified random sampling. However, the results show that all the categories of HCWs had enough participants for analysis. Reviewer Comment: \"Descriptive analysis (time, place, and person) provided preliminary insight into the clinical spectrum and course of COVID-19 among health workers.\"- can't find these in the results.\" Author's Response: We are grateful to you for pointing this out. It had been done as a part of the primary project; however not included in the current manuscript due to the word restriction and to make the finding crisp. We may provide those results in supplementary files if required. Reviewer Comment: \"Couldn't understand why All tables & figures \"n\" variable. table 5 \"Aerosolizing Procedure\" p-value & odds ratio doesn't correlate.\" Author's Response: We are thankful for pointing this out sir, as the ‘n’ in each table varies with different parameters assessed. As in table 1, the characteristics at the baseline and endline are shown. In table 2, IPC practices were assessed among all the participants enrolled at baseline, i.e. from 817 participants. Each subtotal has been mentioned individually as well for clarity. Further in the association tables, those have the respective exposure have been assessed which again was variable as all the HCWs were not exposed to the same kind of exposure. We are thankful to you sir for pointing out the error in values. The values have been corrected in the results table corrected OR value= 0.647(0.333- 1.258) , p value= 0.199 Reviewer Comment: \"Results are not in line with objectives.\" Author's Response: Dear sir, we humbly decline this suggestion and do not agree with this. Kindly find the objective-wise results as presented in table number To study the adherence to IPC measures followed between different categories of healthcare workers. - results shown in table 1 and 2 To estimate seroconversion rate based on the type of exposure with COVID-19 patients among healthcare workers. - results shown in table 3, 4 and 5 To study the association between seroconversion and IPC practices. -results in table 6 Reviewer Comment: \"The conclusion and results don’t correlate completely.\" Author's Response: Thanks for your valuable input. We considered your suggestion, and the authors have redrafted the conclusion sections to make them clearer, and in line with the results. Revised Conclusion: \"In conclusion, based on the gap in IPC practices amongst the HCWs, it is essential to strengthening IPC practices among healthcare workers through refresher training and supportive supervision. Healthcare workers being a part of the community need to ensure the appropriate use of personal protection to prevent contracting any disease and transmitting it to the community and vice versa. In view of the new knowledge added, rational and appropriate use of PPE and other disinfection practices needs to be communicated to the larger audience. Further research on the transmission of COVID-19 in low- and middle-income settings is required.\" I sincerely hope our efforts in improving the manuscript have clarified your concerns and improved the manuscript's quality. We are requesting you to provide your valuable comments on the same. Thanks and Regards"
}
]
}
] | 1
|
https://f1000research.com/articles/11-728
|
https://f1000research.com/articles/11-932/v1
|
15 Aug 22
|
{
"type": "Research Article",
"title": "Penta-Helix Model of E-Government in Combating Corruption in Indonesia and Malaysia: Religiosity as a Moderating Role",
"authors": [
"Pupung Purnamasari",
"Noor Afza Amran",
"A. Harits Nu'man",
"Rusman Frendika",
"Mohamad Naimi Mohamad Nor",
"Mohamad Sharofi Ismail",
"Noor Afza Amran",
"A. Harits Nu'man",
"Rusman Frendika",
"Mohamad Naimi Mohamad Nor",
"Mohamad Sharofi Ismail"
],
"abstract": "Background: E-government is an initiative taken by governments worldwide to align the administration of their countries. Governments have utilized the internet as part of a transition into a globalized economy. This helps reduce red tape and procedures in dealing with people in government agencies. This study aims to develop an e-government model as an anti-corruption strategy by applying the Penta-helix model and religiosity as the moderating variable. Methods: The data was gathered from government officials, representatives in business, media, academia, and NGOs, in Indonesia and Malaysia in 2021. Online questionnaires were distributed to 240 respondents from Indonesia and Malaysia. In addition, SPSS v.25 and SEM AMOS v.25 were used to analyze the data. Results: The findings indicate that the Penta-helix elements and religiosity could help to reduce corruption in Indonesia. Meanwhile, Malaysia must increase its human resource competency and embed the religiosity element as a tool to reduce corruption. Conclusion: Penta-helix and religious factors should be incorporated by organizations in Malaysia and Indonesia as part of their strategy in combating corruption.",
"keywords": [
"Anti-corruption",
"E-Government",
"Information technology",
"Penta-helix",
"Religiosity"
],
"content": "Introduction\n\nThe development of communication and information technology is influential in increasing efficiency or productivity in the business world. In addition, both technologies can improve people’s standard of living globally.1 Governments in several countries have used technology and information to deliver information on government services to the public efficiently. Malaysia has moved on its e-government journey since 1996 with the introduction of the Multimedia Super Corridor (MSC). MSC is an ICT hub hosting more than 900 multinationals, foreign and home-grown Malaysian companies focused on multimedia and communications products, solutions, services, research and development.2 With rapid development and challenges in the digital era, the Malaysian Government created the new Malaysian Digital Economy Blueprint in October 2019 to create a trusted, secure, and ethical digital environment. In addition, the COVID-19 pandemic has accelerated the changes that make the government, businesses, and communities better able to adapt to new ways of daily activities.\n\nFor example, Malaysia, under the Malaysian Administrative, Modernization, and Management Planning Unit, launched the eKL project in 2007. eKL seeks to develop public services through an integrated and connected Klang Valley. Based on the theme One Government—Many Agencies, the eKL project sought to integrate service delivery across agencies in order to ensure that services are delivered in a standardized, systematic, and seamless manner. Under eKL, a number of innovations have been introduced. MyBayar is the online payment gateway that offers citizens a convenient and secure way to make online payments to the government. MyForms is the centralized forms directory that makes forms available to citizens and businesses with downloadable and online submission options. In addition, MySMS15888, the short messaging system, is another channel that enables people on the move to stay connected to government services. It provides two-way communication between government agencies and citizens where governmental information, news, and services are made available to mobile phone subscribers anytime and anywhere.\n\nE-governance is commonly defined as an information and technology application in providing information as well as connecting citizens with governments. The introduction of e-government is an initiative to streamline the administration of a country. It will make government affairs easier and faster, improving the relationship between people, the private sector, and the government. The government’s digitalization system covers various aspects to ensure the effectiveness of the government’s service delivery system. This includes services related to transportation, immigration, health, and education. This digitalization will reduce over-the-counter services and encourage the use of the internet as a means of delivering government services. Thus, location and time are no longer obstacles for these parties to perform their business. In addition, the global use of technology and the reliability of data in the public service will make it easier for citizens to act and facilitate decision-making. In addition, the reliability of government services will improve relations between the two parties.\n\nFurthermore, e-governments function as a tool in reducing corruption incidences and also create higher transparency within the workplace. Consequently, human resources can be optimized and government financial savings are achieved. In addition, the widespread use of technology reduces bureaucracy and human intervention in decision-making. Works will become faster and simpler. However, the smoothness of e-government services will take some time. Government servants and the public must be provided with adequate exposure to adapt to e-government and the e-government system also requires several phases before the system stabilizes.\n\nThe Penta-helix model is used in the study of e-government and corruption. The Penta-helix model is a conceptual framework that involves academics, government, industry, non-governmental organizations, and civil society/entrepreneurs/media. It facilitates economic development and pursues innovation and entrepreneurship through collaboration and synergy.3–6 The interactions between the components in the Penta-helix model facilitate new knowledge and innovation and also create innovation-based economic dynamics.7 Various studies have examined the connection between corruption and e-government. Research on the effect of e-government on corruption highlights that a gap remains in terms of theory and research method that requires further exploration. Several studies on the effect of e-government on corruption were case studies, while others used empirical methods at the macro-level.8 Furthermore, several previous studies have examined the e-government effect on corruption, but without considering other determinants such as political, social, and technological factors.9 Furthermore, the Penta-helix model will help reduce inhibiting factors in implementing the licensing bureaucracy.5 It is practical for problem areas of multi-stakeholders, where they represent a range of a site or problem’s interests.10\n\nThis study focuses on developing a model of e-government for fighting corruption in Indonesia and Malaysia by utilizing the penta-helix model, which focuses on the synergy between stakeholders in organizational innovation11 and religiosity.12 Consequently, it is expected that a comprehensive and inclusive e-government model, providing services to the public and empowering public participation in eradicating corruption, will be established. This study selects Indonesia and Malaysia because both are ASEAN countries with similar cultures, beliefs, and understanding.13 In addition, the corruption level in both countries is alarming.14\n\nThis study was conducted for several reasons. First, the e-government’s application in developing countries involves various challenges.15 Corruption occurs when there is an abuse of power and a lack of religious values.12,16 Pressure, opportunity, and rationalization can also result in corruption.17 Based on previous studies, it is crucial that this study be undertaken in Indonesia and Malaysia to reveal and share new findings.\n\nFirstly, the governments of both countries have not succeeded in suppressing corruption, which is not worth the cost of eradicating corruption. Furthermore, the ranking of Transparency International highlights that the countries’ positions do not significantly change yearly.18 Therefore, there is a need for e-government transparency through the publication of data on government actions and decisions, the existence of mechanisms to protect whistle-blowers, the maximum punishments for corruptors, and the implementation of information communication and technology.\n\nSecondly, the current system lacks the impact of religiosity and the lack of supervision through the penta-helix dimension, making corruption more crucial. Finally, in practice, e-government is a vulnerable platform and prone to hacking, such as in the e-procurement of goods and services.\n\n\nLiterature review\n\nE-government implementation in Malaysia began seriously in 1996. In that year, the Multimedia Super Corridor (MSC) project was launched by the government, and e-government was one of the essential components of the flagship application introduced in the MSC. The e-government implementation is intended to increase the community’s productivity and public services quality. In addition, an introduction about e-government will enable people to access services faster, increase competitiveness, and reduce the digital divide in society.\n\nA study by Ramli19 found that as far as e-government implementation challenges are concerned, several enhancements require consideration. For example, there is a low infrastructure technical quality, unstable connectivity, slow speed connection, and limited internet access in certain rural areas. In terms of the structure of legislative, Malaysia has restrictive laws and regulations, outdated regulations, redundant acts, and both security and privacy concerns. Furthermore, Malaysia faced financial constraints of insufficient funds and the necessity on improving the partnership on the public-private. In terms of human infrastructure, it was found that Malaysia did not have an individualistic culture.\n\nMeanwhile, citizens and public officials are slow in adapting to changes. The officials’ public are having difficulty changing in the initial implementation phase, and lack skills as well as expertise, little motivation, and changing management of training. In addition, there is limited integration and coordination, and the issues of federal-state power slow down the implementation of the pace. Issues such as accountability, transparency, and corruption are always associated with the procurement system in Malaysia. This is a major concern of the stakeholders, especially regarding mismanagement and wastage of public money. Contractors and stakeholders demand transparency in the procurement system. Selecting succeeded contractors based on cronyism and personal relationships rather than professional evaluation has become a serious phenomenon. The procurement officers and tender boards were blamed for non-compliance and malpractice with procurement procedures and policies.20\n\nHamzah et al., found that government bureaucracies have created barriers to government services and caused poor accountability and transparency, thus, discouraging citizens’ dealings with government agencies.21 Therefore, society must change and be open to technology. The e-government must quickly implemented so that the citizens and government can make a connection to the virtual space that created and strengthened through the leadership that support governance framework.21 The government acknowledges that technology enables various social problems, including corruption, to be addressed and will continue to find the best mechanisms by using the latest technologies to combat corruption. As stated by Ramli, a positive connection between corruption and e-government when society reaches high level of economic growth and development.22 This is also supported by Rustiarini and Sudiartana, where different organizational and external factors change the e-government functions’ effectiveness in fighting corruption.23\n\nIn Malaysia, the National Anti-Corruption Plan (NACP) 2019–2023 states that technology can be used to prevent corruption where complaints are obtained through open public sources with transparent processes. The electronic process has seen procurement and payments activities run more transparently through digital transactions, reducing the risk of corruption.24 The latest technology, which indirectly creates a safer audit trail, is more reliable and trusted by stakeholders. In addition, information technology facilitates access to information and promotes transparency and openness that contribute to fair and equitable competition for the government and the market. Therefore, through the digitalization of the civil service, bureaucracy and the risk of corruption and government costs can be reduced.24\n\nFor Indonesia, attempts have been made to eradicate corruption. The Indonesian government focuses more on reporting the application of electronic-based state finances, starting from local governments to the central government and maximizing the institution of an independent corruption eradication commission. In addition, the implementation of e-audit in financial audit institutions aims to examine the use of state finances more quickly and accurately. Therefore, implementing e-government and e-audit in every part of the government office can reduce acts of fraud and corruption in government institutions.\n\nOne of the strategies to prevent acts of corruption is to implement e-government. A study by Alhammadi and Alhadramy highlights the actions to prevent corruption through the implementation of e-government which includes25:\n\na) The use of services undertaken online will increase efficiency and reduce discretionary abuse and other corruption’s opportunities.\n\nb) The function of e-government developed through the government must be complemented by the development of institutions, laws, and practices that protect whistle-blowers and provide disincentives and laws against perpetrators of corruption.\n\nc) E-government will reduce discretionary power and control the operations of government officials. In addition, activities undertaken by the public will be tracked, controlled, and stored for future reference.\n\nd) E-government tools track various illegal events and actions and proactively detect suspicious behavior before any crime is committed. Thus, officials are more careful in trying to engage in corrupt behaviors since transactions are documented and easily traceable.\n\ne) Government information is available online. Citizens can simply access and connect to the government services and information anywhere, anytime, and through various channels. Thus, it helps citizens provide proper documentation when lodging their complaints against corrupt practices.\n\nThe penta-helix model is often used in fields related to socio-economic development. The model contains five important elements namely (i) public, (ii) private, (iii) academia, (iv) society, and (v) entrepreneurs. These five elements support the achievement of the economic development level. They have roles and functions in the development of society. The penta-helix model helps to improve the economy through innovation and entrepreneurship, and collaboration and synergy among the players.3–6 They are agents of local economic change and promote sustainability in economic activities. Therefore, the economic development of an area will be determined by their commitment and contribution.\n\nIn achieving the socio-economic development goals, these five parties must ensure that the goals are running smoothly. Therefore, close cooperation and understanding must be established. The importance of a synergistic relationship between the five components in the penta-helix model is vital, where the interaction between these components will result in innovation and knowledge and create dynamics of an innovation-based economy.8,26 Such synergistic relationships include ensuring that economic activities are conducted ethically by avoiding activities that contribute to elements of corruption in business. This is because these elements are interdependent in the business ecosystem, and corrupt activities can be avoided if each of these elements acts seriously to address them. Therefore, each key player must have a sense of responsibility and work together so that the relationship established contributes to fair and equitable economic development.\n\nFraud is defined in various ways. However, the focus is a person’s deceitful behavior that is designed to benefit oneself by misrepresentation.27 A fraudulent financial statement is a designed act that impacts in a misstatement material in the financial statements. Meanwhile, Sihombing and Rahardj28 said that fraudulent financial reports are intentionally produced in financial statements that are not in conformance with commonly accepted principles of accounting. Fraudulent reports are material and can influence the decisions of attentive parties. Compatible with the Australian Audit Standard in US and European, a fraudulence financial report is an premeditated misstatement, as well as the omission of amounts or a leak in financial statements to mislead the users of these statements.29\n\nThe theory of fraud was first expressed by Skousen et al.,17 which consisted of insurmountable financial issues, opportunities to commit violations and rationalization by offenders. Over time, Cressey’s hypothesis developed rapidly and became known as “the fraud triangle”. Details about the fraud triangle are depicted in Figure 1.\n\nIndividual behavior patterns are affected by a set of values consisting of intrinsic and extrinsic values. One aspect of these values is the religious value.30 Religious values are those that inspire self-confidence and faith in an individual. These values guide an individual to act obediently and honestly in dealing with daily activities.31 A person’s religious values can affect self-control and reduce deviant behavior.32,33 A study found that religiosity positively correlates with risk-averse actions. In fact, in the case of some religions and religious people tend to avoid risky actions such as corruption.34\n\nHowever, there is another perception of corruption based on religiosity and social trust. The view rests on the belief that a higher divine power will punish individuals for their evil deeds in this lifetime or next. With these beliefs, people will not engage in corrupt behavior, and there is no need to monitor the corrupt behaviors of others,35,36 including in government institutions.37 In addition, trust in other people and law enforcement systems may reduce the demand for accountability of actions. Furthermore, fewer actions are required to monitor the elected officials by having more religious government officials.38\n\nA person’s level of faith can indirectly influence a politician into political corruption. Religious assistance is shown both empirically and theoretically to increase political aid39 and civic commitment.40,41 However, the motives behind such commitment could be due to rent-seeking, which increases political corruption. In addition, there are other economic motivations for those who are religious: religion can add to an individual’s moral character, as a principal value in transaction of the marketplace.42 Futhermore, people in the same organization can be more tolerant of corrupt behavior carried out by others who are part of the same organization’.43\n\nInvestigations of the relationship between religiosity and corruption hace found different results.44 Research in the United States shows that there is no relationship between the level of corruption and religiosity.44 Futhermore, in tests done globally, a negative relationship between the level of corruption and religiosity has been founf.45 However, this study did not control certain standard variables associated with corruption, such as per-capita GDP, cultural colonialism or the effectiveness of the legal system. A study by Yahya et al.,46 found that employee religiosity and organizational culture are essential in determining the corruption millennial employment. This finding suggests that it is important for management to emphasize the importance of a positive inconclusive organizational culture and religious awareness among employees. A study by Barbier et al.,47 found a negative relationship between individuals’ religiosity and their responses to what is “justifiability of corruption,” thus, suggesting there is no aggregate government corruption.\n\nCorruption is detrimental to the overall well-being of a country’s economic system. While legal enforcement systems are available in the majority of countries, corruption still exists. Many researchers have analyzed the factors that correlate with corruption in different countrie and one of the main factors that influence the level of corruption is religiosity.48 However, previous research has had very little explicit focus on the difference between religion and religiosity. A study by Gokcekus and Ekici uses data from various countries and experiments with varying measures of religiosity.48 The empirical proof indicates that religiosity, rather than a religious affiliation, influences corruption levels. Societies that are more religious typically have higher levels of corruption, despite the country’s religious affiliation.\n\n\nHypothesis development\n\nThe primary cause of corruption in the government sector is the asymmetry of information between the government and public. Consequently, this can result in bureaucrats setting ambiguous standards of government service. This facilitates bribery through secret contacts with the public, and the public is more likely to respond to the agreement.49 E-government can reduce corruption because it can control corruption through transparency,50 disclosure, and the monitoring of government administration using technology.51,52 Research reveals that e-government is beneficial in reducing corruption.53–57 In terms of the use of e-government in the community, more attention is given to the transparency, objectivity, publication of government budget information, and feedback related to services and policies.58 The solutions offered by the e-government expect sophistication from a technical perspective. Furthermore, they require a complete reorientation of the bureaucracy, especially, the awareness to conduct duties and functions to be neutral and genuinely in the mission of public service.58\n\nTherefore, e-government is essential in preventing corruption if the maturity of e-government implementation involves public participation in information, public participation in supervising the government, and actions from government officials that substantially increase transparency.60 Furthermore, the synergy between academics, businesses, the community, media, NGOs, and governments are elements of the penta-helix. Therefore, this study hypothesized that:\n\nH1: E-governance has a positive effect on corruption.\n\nPeople with intrinsic religiosity can be defined as people having reached a certain level of personal integrity and maturity. They have made an unconditional commitment toward religion and make decisions independently. Religious orientation becomes an inseparable part of one’s religious maturity.31 Specifically, religiosity refers to a person’s belief, practice, experience, identity, and attitude.61 For Muslims, religiosity means adherence to God and desire to be a good person.16\n\nResearch by McGee et al.,62 and Shadabi63 has highlighted that religion could influence human behavior and individual decision making. A study found that religiosity can moderate the relationship between corruption and e-government because a person’s reaction is dependant on their beliefs, values and responsibilities.31 In addition, Said et al.,64 identified that religiosity is significantly give impact to asset misappropriation. Higher religiosity in a person lowers the probability of asset misappropriation. Therefore, individuals with a high level of religiosity would attempt to avoid deviant actions.65 Therefore, a religious background and knowledge instilled in an individual can help reduce their level of corruption because they can control themselves in facing normal situations and irregularities within the workplace.32 Hence, religiosity can prevent individuals from committing fraud and corruption.31 Therefore, based on previous studies, this study hypothesized that:\n\nH2: E-government has a positive effect on corruption moderated by religiosity.\n\nThe penta-helix model is used in this diagram to predict the forms, causes, and efforts that prevent corruption. This study involved five group of participants from both the Indonesian and Malaysian Governments, businesses, community, academia, and the media. The moderating of religiosity highlights how humans behave and comply with rules and regulations. This determines how people react when facing the risk of corruption and fraud.59\n\nThe fraud triangle theory in the diagram indicates that “rationalization” is a perspective related to moral and psychological elements, which are essential in identifying the causes unethical behavior.66 Religiosity can be a “zero tolerance” policy toward unethical behavior and can be a prevenative factor when they consider undertaking unethical behavior.\n\n\nMethods\n\nThis study was accepted and approved by the Universitas Islam Bandung Ethical Clearance Committee (no. 719/B.04/Bak-k/XII/2020). In addition, written, informed consent was obtained from participants. The variable utilized in this study is e-government which is divided into three sections (3) –the Government to the Citizen (G2C), the Government to the Business (G2B), and the Government to the Government (G2G). Meanwhile, the penta-helix was measured by elements such as government officials, academia, media, NGOs, the community, and businesspeople. The religiosity variable was measured based on ideology, practice, experience, knowledge, and consequences. Finally, corruption was measured using opportunity, pressure, and rationalization. The variables are operationalized in Table 1:\n\n\n\n- Clarity\n\n- On-time\n\n- Easiness\n\n\n\n- Availability\n\n- Ownership\n\n\n\n- Solve problem\n\n- Accessible\n\n- Feedback\n\n\n\n- Involvement\n\n- Role\n\n- Participation\n\n\n\n- Clarity\n\n- On-time\n\n- Easiness\n\n\n\n- Fair competition\n\n- E-procurement\n\n\n\n- Clarity\n\n- On-time\n\n- Easiness\n\n\n\n- Function\n\n- Action\n\n- Evaluation\n\n\n\n- Clarity\n\n- On-time\n\n- Speed\n\n\n\n- Role\n\n- Involvement\n\n- Participation\n\n\n\n- Clarity\n\n- On-time\n\n- Easiness\n\n\n\n- Educate\n\n- Controlling\n\n- Supervising\n\n\n\n- Clarity\n\n- On-time\n\n- Easiness\n\n\n\n- Belief\n\n\n\n- Clarity\n\n- Easiness\n\n\n\n- Worship\n\n- Convenience\n\n\n\n- Clarity\n\n- Easiness\n\n\n\n- Activity\n\n\n\n- Clarity\n\n- Easiness\n\n\n\n- Law understanding\n\n\n\n- Clarity\n\n- Easiness\n\n\n\n- Obedience\n\n- Responsibility\n\n\n\n- Clarity\n\n- Easiness\n\n\n\n- Report\n\n\n\n- Clarity\n\n- Firmness\n\n\n\n- Checking\n\n\n\n- Available\n\n- Intention\n\nThis study utilized a sample of 240 respondents from Malaysia and Indonesia between March to May 2021. These countries were selected because they share similar cultures and beliefs. In addition, the corruption level in both countries is alarming. The respondents represented those in the petha-helix model: government officials, academics, those working in the media, NGOs, the community, and business people, in Malaysia and Indonesia. Respondents included government auditors, university lecturers, by news employees, NGO employees in the social sector, community employees in the economic and social sector; and business people at the Ministry of Micro, Small and Medium Enterprises (MSME) level. The sample was selected based on purposive sampling.69 The respondent selection criteria was based on their job description and experience or daily use of e-government services. Respondents who had never used e-government were not included in the sample criteria. The respondents were contacted by the research team, using established networks of friends, office colleagues, families, and alumnus to recruit participants. The researchers then approached the respondents through telephone calls, email, or WhatsApp messages. After explaining the aims of the study to the respondents and receiving their consent, a questionnaire on Google Forms was sent either through email or WhatsApp.\n\nThis study used a quantitative approach for the data collection procedure. Before this questionnaire was distributed to respondents, it was tested on 30 respondents, using a cross sectional method through focus group discussions in February 2021. Based on this pilot project of 30 respondents, the results showed that the instrument the questionnaire was valid and reliable.\n\nAfter receiving comments and refining the questions from the 30 respondens in the pilot study, the revised questionnaire was distributed to 240 new respondents via emails and WhatsApp message using the Google Form platform, between March to May 2021. The questionnaire was divided into five sections. Section A: respondent profile; Section B: The implementation of e-government in local authority; Section C: perspectives related to fraud triangle dimensions; Section D: beliefs and religiosity; and Section E: strategy to combat corruption.\n\nThe descriptive and inferential non-parametric statistics (part of inferential statistics) were used in analyzing the data. The SPSS v.25 was used to analyze the data for Malaysia and Indonesia datasets. Subsequently, this study conducted a reliability and validity analysis to assure the stability of the data. The estimated r-value was compared to the tabled r-value in determining the validity value. The validity test was deemed passed if the calculated r-value was larger than the tabled r-value. While the reliability test was conducted using Cronbach’s Alpha with criteria ranging from 0.80 to 0.88.\n\n\nResults\n\nFour demographic information were gathered from the 240 respondents from Malaysia and Indonesia— gender, education, computer abilities, and daily computer usage. First, there were more female than male participants from both countries in this study (Indonesia = 53%; Malaysia = 50%). Second, the average age is 36–45 years old (37% for Indonesia and 47% for Malaysia). Third, the respondents’ average educational level is an undergraduate degree (43% in Malaysia and 42.5% in Indonesia). In addition, this study shows no difference in terms of computer skills between Indonesia (79%) and Malaysia (80%). Finally, the average daily computer usage is six (6) hours per day (36% in Indonesia and 47.5% in Malaysia). Respondents’ complete information of demographic is displayed in Table 2:\n\nThe results from the test of validity and reliability show that all items have an r-count value larger than the interpolated r-table (n = 240, = 5% = 0.133). All items are declared valid and reliable based on the validity and reliability testing results. Furthermore, the variables analyzed have a positive and notable effect for Indonesia and Malaysia since the p-value is below 0.133, except for the religiosity variable, which is not too significant because larger p-value (0.162), as shown in Table 3.\n\nTable 4 highlights that the effect of e-government on corruption in Indonesia is 0.132, whereas the effect of religiosity on corruption is negative (−0.276). Nevertheless, the presence of religiosity as a moderator can boost the effect of the e-government on the corruption by 0.321. However, the effect of the e-government on the corruption in Malaysia is relatively minimal at 0.066—religiosity is only 0.053, and religion as a moderator is 0.209. Therefore, the impact of religiosity toward e-government corruption is minimal. Therefore, in comparing the moderating effect of religiosity in both countries, it appears that religiosity significantly strengthens the influence of the e-government on the corruption in Indonesia more effectively than in Malaysia. The following graph depicts the number of e-governments on corruption in Indonesia and Malaysia and its moderating effect. Interestingly, these findings provide evidence that religiosity is influential in combating acts of corruption in government agencies. Thus, the regulatory bodies and the governments of both countries must be aware that religiosity may influence the behavior of the officials.\n\n\nDiscussion\n\nThe results of Indonesia’s e-government rollout reveal that e-government implementation had an effective and significant effect on reducing corruption, as indicated in Table 4 (0.132). According to Sosiawan,70 e-government deployment is considered a “dare-dare” project or merely a fleeting trend. Therefore, all government policies, legislations, and practices, including the use of e-government, impact corruption. This implies that public transparency is required. This finding supports Nam’s50 assertion that e-government can minimize corruption since it has the potential to control it through openness. This conclusion contradicts the findings that claim that the e-government negatively impacts corruption.53–57,60\n\nThe findings suggest that the majority of e-government providers, including government and non-government organizations, are still “secure” and “comfortable” with website owners and are no longer concerned with optimizing e-government use. However, there are hints that e-government is simply a project to “sell” communication and information technologies, both hardware and software, to “traders.” This indicates that one’s beliefs and religion do not become a barrier against acts of corruption, but that religion is utilized as a tool and justification for corruption by an official. According to the findings, based on the penta-helix model, government involvement can help create a clean, committed, and high-integrity work environment. NGOs can promote improvements in government officials’ behavior and the elimination of official corruption. Therefore, while academia may help analyze research data and facts for the government to consider when making policies and decisions, data and news can be communicated to the public in a timely, accurate, and transparent manner, and businesspeople can follow and maintain good business ethics in compliance with the laws. Meanwhile, religion is a moderator that can boost the influence of e-government on corruption (0.321). It can positively reduce the acts of corruption, particularly the religiosity variable, which will become more important in reducing corruption in Indonesia. E-government within Malaysia shows a positive impact on both corruption (0.066) and religiosity (0.053), as displayed in Table 4. The results also highlight that religiosity moderates (0.209) and strengthens the impact of e-government on corruption in Malaysia.\n\nIn summary, using the penta-helix model, the NGOs, government, academia, businesspeople, media, and the community can be leveraged to boost anti-corruption efforts. Similarly, religious qualities such as belief, kindness, and trust can support anti-corruption efforts.66 The result on religiosity aligns with previous studies, showing that religiosity can strengthen the efforts to eradicate corruption.31,65 Thus, religion can be employed as a moderating variable in both Indonesia and Malaysia to improve the influence of e-government on corruption, reduce pressure from other parties, close the chances for corruption, and reduce the problem of rationalization.\n\nThere are four quadrants in Figure 4, representing four situations—low priority, unexpected, priority, and achievement. In Indonesia, there are still many cases of bribery in project tenders, unscrupulous officials marking up project values, businesspeople and officials flirting with the acquisition of government projects, the greedy nature of unscrupulous officials and businesspeople, and a lack of fear of God for their actions. Therefore, G2B conditions in Indonesia are higher (0.639) than in Malaysia (0.504). Meanwhile, in Malaysia, the cases of bribes related to procurement are still high, as reported in the National Anti-Corruption Plan. However, the state of G2C in Indonesia is lower (0.625) than in Malaysia (0.758) due to the lack of community initiative and apathy in supervising government and industry projects, and boredom with government promises to eradicate corruption that has never been fulfilled.64 Both countries reported a high value for G2G, which is above the average score in attempts to eradicate corruption. However, based on the G2G implementation (Indonesia, 0.650; Malaysia, 0.745), Malaysia’s level of eradication is significantly greater than Indonesia’s.\n\nConsequently, both countries aim to have a high level of corruption prevention and eradication by fostering good governance and growing G2B and G2C engagements. For both countries, the influence of e-government adjusted by religion has a favorable and significant impact. According to the laws, anyone with a high level of religiosity can ensure that e-government projects are completed on-time while avoiding corruption. However, because poor religious comprehension is reflected in daily actions, weak faith is readily seduced to commit corruption, worship obedience can be a ruse, and religious regulations that should be understood are ignored.\n\nTherefore, the e-government system is a strategy that can be used in Indonesia and Malaysia to prevent corruption. Strengthening the understanding of religious beliefs and elements of penta-helix will help both countries to have better transparency and a strong governance system. With reference to the penta-helix model, the government officials’ strategy is to react truthfully and be transparent in their everyday operations. The role of the media is to be transparent by sharing the latest news and activities of influential people or political elites involved in corruption. Meanwhile, businesspeople aim to encourage business actors to use the e-government platform to perform government-related commercial activities. The role of the NGOs is to oversee the monitoring and the implementation of the e-government process. Meanwhile, academia is encouraged to conduct further research on the prevention of corruption. The most important element is to encourage government officials to improve and increase their religiosity to strengthen their faith. Consequently, they will be less likely to commit corruption.\n\n\nConclusion\n\nThis study found that the implementation of the e-government has positive and significant effects on efforts to prevent corruption in Indonesia and Malaysia. In addition, it proved that religiosity is essential in increasing the influence of e-government in preventing corruption by including elements of the penta-helix. The findings confirmed the objective of the study that penta-helix and religiosity are vital elements to be applied in the government of both countries, especially in the e-government settings. However, this study is limited in that it only considers two ASEAN countries. Therefore, future studies may consider a more extensive dataset which may include other ASEAN countries such as Singapore, Thailand, and Brunei. Moreover, this study only considers the fraud triangle theory. However, further research can apply the “law and regulations” variables, as mentioned in the development of the penta-helix model, called the hexa-helix model, so as to produce stronger results.\n\n\nData availability\n\nFigshare: Dataset of Questionnaire Result from the respondents of Penta-helix model of e-government in combating corruption\n\nhttps://doi.org/10.6084/m9.figshare.19643325.v171\n\nThis project contains the following underlying data:\n\n• Dataset of Questionnaire Result from the respondents of Penta-helix.csv\n\nFigshare: List of questions and descriptions of questionnaire of the Penta-helix model of e-government in combating corruption in emerging markets: Religiosity as a moderating https://doi.org/10.6084/m9.figshare.19643607.v172\n\nThis project contains the following extended data:\n\n• List of questions and descriptions of questionnaire of the penta-helix model.csv\n\nFigshare: The Respondent characteristics of the penta-helix model of e-government in combating corruption in emerging markets: Religiosity as a moderating role\n\nhttps://doi.org/10.6084/m9.figshare.19643340.v173\n\nThis project contains the following extended data:\n\n• The Respondent characteristics of the penta-helix model of e-government.csv\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).",
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Indonesia:2022.\n\nPurnamasari P, et al.: List of questions and descriptions of questionnaire of the Penta-helix model of e-government in combatting corruption in emerging markets: Religiosity as a moderating, Figshare. Indonesia:Figshare;2022.\n\nPurnamasari P, et al.: The Respondent characteristics of the Penta-helix model of e-government in combatting corruption in emerging markets: Religiosity as a moderating role, Figshare. Indonesia:Figshare;2022."
}
|
[
{
"id": "147536",
"date": "05 Sep 2022",
"name": "Hussaini Bala",
"expertise": [
"Reviewer Expertise Financial reporting",
"auditing",
"corporate governance",
"sustainability reporting",
"and taxation",
"financial management",
"economic growth"
],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address 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 done well by addressing an interesting topic ‘Penta-Helix Model of E-Government in Combating Corruption in Indonesia and Malaysia: Religiosity as a Moderating Role. However, I have the following comments that can be used to enhance the quality of the manuscript.\nMajor Comments\nThe focus of the paper is corruption in Indonesia and Malaysia. However, the introduction majorly concentrates on E-Government. Therefore, there is need for the authors to highlight the prevalence of corruption in Indonesia and Malaysia and how E-Government may help in combating the corruption.\n\nThere is need to amend the research framework to reflect the title of the paper. This is because the current framework in the paper displays E-government as a dependent variable.\nMinor comments\nYou may amend the topic to ‘Penta-Helix Model of E-Government in Combating Corruption in Indonesia and Malaysia: The Moderating effect of Religiosity.'\n\nIn the literature review section, change the title ‘E-government corruption effect moderated by religiosity’ to ‘E-government, religiosity and corruption.' Or 'Influence of religiosity on the link between E-government and corruption’.'\n\n'H2: E-government has a positive effect on corruption moderated by religiosity' should be changed to ‘H2: Religiosity has a moderating effect on the link between E-government and corruption.'\n\nIs the work 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/11-932
|
https://f1000research.com/articles/11-127/v1
|
01 Feb 22
|
{
"type": "Brief Report",
"title": "Unprecedented early-summer heat stress and forecast of coral bleaching on the Great Barrier Reef, 2021-2022",
"authors": [
"Blake L. Spady",
"William J. Skirving",
"Gang Liu",
"Jacqueline L. De La Cour",
"Cathy J. McDonald",
"Derek P. Manzello",
"William J. Skirving",
"Gang Liu",
"Jacqueline L. De La Cour",
"Cathy J. McDonald",
"Derek P. Manzello"
],
"abstract": "The Great Barrier Reef (GBR) is predicted to undergo its sixth mass coral bleaching event during the Southern Hemisphere summer of 2021-2022. Coral bleaching-level heat stress over the GBR is forecast to start earlier than any previous year in the satellite record (1985-present). The National Oceanic and Atmospheric Administration (NOAA) Coral Reef Watch (CRW) near real-time satellite-based heat stress products were used to investigate early-summer sea surface temperature (SST) and heat stress conditions on the GBR during late 2021. As of 14 December 2021, values of instantaneous heat stress (Coral Bleaching HotSpots) and accumulated heat stress over a 12-week running window (Degree Heating Weeks) on the GBR were unprecedented in the satellite record. Further, 89% of GBR satellite reef pixels for this date in 2021 had a positive seven-day SST trend of greater than 0.2 degrees Celsius/week. Background temperatures (the minimum temperature over the previous 29 days) were alarmingly high, with 87% of GBR reef pixels on 14 December 2021 being greater than the maximum SST over that same 29-day period for any year from 1985-2020. The GBR is starting the 2021-2022 summer season with more accumulated heat than ever before, which could have disastrous consequences for the health, recovery, and future of this critical reef system.",
"keywords": [
"Background temperature",
"Bleaching",
"Coral",
"Degree Heating Week",
"Great Barrier Reef",
"heat stress",
"La Niña",
"satellite monitoring"
],
"content": "Introduction\n\nThe Great Barrier Reef (GBR) has endured five mass coral bleaching events, three of which took place between 2016 and 2020. Coral bleaching occurs when stress disrupts the symbiosis between corals and their endosymbiotic algae (zooxanthellae), causing the corals to expel zooxanthellae; this can lead to coral mortality if stress is prolonged or severe (Brown, 1997). Mass coral bleaching, which is bleaching at a scale of an entire reef system or geographic realm, has only been linked to the stress of excess sea surface temperatures (SST), and this is expected to happen when heat stress in a region exceeds a certain intensity or duration (Glynn, 1984; Hoegh-Guldberg, 1999; Skirving et al., 2019). The U.S. National Oceanic and Atmospheric Administration’s (NOAA) Coral Reef Watch (CRW) program has developed a number of satellite-based SST products that are used to monitor oceanic heat stress, including on coral reefs. The Degree Heating Week (DHW) is an accumulation of instantaneous heat stress (Coral Bleaching HotSpots, or just HotSpots) over a 12-week running window (Skirving et al., 2020). A DHW value of 4 degree Celsius-weeks (C-weeks) or greater is capable of causing sufficient stress for corals to bleach significantly (Hughes et al., 2018; Skirving et al., 2019). In the early-summer months preceding the five documented mass bleaching events, heat stress on the GBR had never exceeded a DHW of 3 degree C-weeks prior to mid-January (with the earliest occurrence in 2002 on 12 January), with peak stress typically occurring between late February and early March. In late 2021, sections of the northern GBR reached a DHW ≥ 3 degree C-weeks by 13 December, and given the observed conditions at the time of writing (21 December 2021), roughly one third of the GBR is expected to exceed a DHW of 4 degree C-weeks by late January 2022, which is unprecedented.\n\nIn addition to the satellite-based measurements, NOAA CRW’s modelled Four-Month Coral Bleaching Heat Stress Outlook product, from as early as 2 November 2021, forecast sufficient heat stress to result in a potential sixth mass coral bleaching event on the GBR during the 2021-2022 summer season. The anticipated mass bleaching event was not only forecast to occur during atypical climatic conditions (i.e., this would be the first mass coral bleaching on the GBR during a La Niña), but was also forecast to be the earliest onset of a summer-time heat stress event for the GBR to date. During the eleven-year period covered by CRW’s Outlook product (2011-2021), the GBR has experienced three of its five known mass bleaching events, one of which (2020) occurred as a result of the most extensive heat stress event the region has suffered (Hughes et al., 2021). Here, we will describe the early-summer SST and heat stress conditions on the GBR in 2021 (November-December), modelled forecasts of heat stress, and how these compare to observations made in previous years, including the relationship between coral bleaching and the El Niño-Southern Oscillation (ENSO) state.\n\n\nResults and discussion\n\nOn 14 December 2021, 59% of the 5 km-resolution satellite-based reef pixels (0.05° × 0.05° satellite pixels that coincide with reefs) on the GBR had HotSpots greater than 0.5 °C, with 34% being greater than 1.0 °C. This is more extensive and severe than the HotSpots measured on 14 December during any year since 1985. The accumulation of HotSpots as DHWs for the same date reached a DHW > 2 degree C-weeks for 14% of GBR reef pixels. DHWs greater than 2 degree C-weeks have accounted for more than 0.1% of the GBR reef pixels by 14 December during only three other years (2008: 1.2%, 2010: 10.8%, 2018: 5.4%), none of which were as high as by 14 December 2021 (13.7%, Table 1). The seven-day SST trend as of 14 December 2021, indicated that SST had increased by at least 0.2 degrees C/week for 89% of GBR reef pixels, with 75% greater than 0.5 degrees C/week. The extent and magnitude of these SST trends are not atypical, and were greater during the same period for several other years, notably 1990 and 1993. However, compared to previous years, the early-summer combination of increasing SST trends with unprecedented levels of extensive HotSpots and DHWs are priming the GBR region for coral bleaching-level DHWs later during the 2021-2022 summer.\n\nFor each year, as of 14 December, the El Niño Southern Oscillation (ENSO) status (October-December) including the NINO3.4 temperature (degrees C); if there was an occurrence of a mass bleaching event on the Great Barrier Reef (GBR) during the following summer; the percentage of the 5,274 GBR 5 km satellite pixels that had a HotSpot > 1; the percentage of GBR pixels with a Degree Heating Week (DHW) > 2; and the mean background temperature for all GBR pixels. Cells within the table containing ‘--’ indicate a value of between 0.0% to 0.1% of the GBR pixels. [Note: La Niña conditions present in December 2016 did not persist past January 2017; bleaching did not commence until February 2017.]\n\nIt is possible that the underlying catalyst for the developing anomalous SST conditions lies in the background temperature. In this context, we define the background temperature as the minimum temperature during the previous 29 days (period of a tidal cycle). The background temperatures on the GBR for 14 December 2021 were overwhelmingly the highest across the entire satellite SST record (i.e., compared with the minimum temperature for each pixel within the 29 days prior to and including 14 December of all years from 1985 to 2020). In fact, for 87% of GBR reef pixels, the minimum temperature at each of these pixels from 16 November to 14 December 2021 was greater than the maximum temperature at the corresponding pixel for any day between 16 November and 14 December from 1985 to 2020 (Figure 1). This is even more noteworthy considering that during November 2021, Queensland, Australia experienced rainfall 136% above the 1961-1990 average, making it the wettest (and most likely the most intensively cloud covered with minimal surface solar heating) November since 2010 (Australian Bureau of Meteorology). Despite this, the SST and corresponding heat stress conditions on the GBR by mid-December 2021 exceeded, in intensity and extent, that seen for any prior year, including bleaching years (Table 1). This suggests that excessive heat energy is being caused by sources other than direct solar heating. Annual SST trends will need to be considered to understand why early-summer 2021 stands out as the warmest on record for the GBR (Figure 2).\n\nMap of Great Barrier Reef 5 km-resolution satellite pixels (0.05° × 0.05°) denoting the difference between the background temperature for 14 December 2021 (minimum SST observed over the previous 29 days, 16 November - 14 December) and the all-time (1985-2020) maximum temperature between 16 November and 14 December. Values indicate difference in degrees Celsius; positive values and warm colours indicate reef pixels in which the 2021 background temperature is higher than the all-time maximum.\n\nDaily SST (degrees Celsius) from 1985-2021 for a single 5 km-resolution satellite pixel (0.05° × 0.05°) located in the northern Great Barrier Reef, Raine Island (11.589°S, 144.035°E). SST for 2021 is in black and in bold.\n\nThe NOAA CRW modelled weekly global Four-Month Coral Bleaching Heat Stress Outlook product has been generating weekly forecasts of bleaching-level heat stress conducive to mass coral bleaching since July 2011 (Eakin et al., 2012; Liu et al., 2018). Using SST forecasts from the NOAA/National Weather Service/National Centers for Environmental Prediction’s Climate Forecast System Version 2 (CFSv2) (Saha et al., 2014), the CRW Outlook product predicts the likelihood of coral bleaching-level heat stress, on subseasonal-to-seasonal scales, up to four months into the future. The CFSv2 is an operational, dynamical, fully coupled ocean-land-atmosphere seasonal climate global forecast model system. CRW’s CFS-based Four-Month Coral Bleaching Outlook is detailed in Eakin et al. (2012) and Liu et al. (2018). Weekly Outlooks generate forecasts of heat stress (HotSpot and DHW) for each of the subsequent 20 weeks at a spatial resolution of 0.5° × 0.5° (approximately 50 km × 50 km). The Outlook product forecasts each 50 km ocean pixel to be in one of five stress level categories, with a corresponding potential bleaching intensity, listed in Table 2. Note that the Outlook system applies a much more complicated algorithm (described in Liu et al., 2018) based on the relationship between CRW’s satellite HotSpots and DHWs as well as modelled HotSpots and DHWs. Forecasts are available in ten pre-set probabilistic levels ranging from 10% to 100% in increments of 10%. Henceforth, when referring to Outlook forecasts, we are referring to the 90% probabilistic Outlook level.\n\n‘Definition for satellite monitoring’ column details the conditions necessary to meet each Stress Level category. The ‘Potential bleaching intensity’ column details the expected outcome for corals subject to each Stress Level.\n\nOn 14 December 2021, NOAA CRW’s near real-time satellite monitoring indicated that nearly the entirety of the GBR (98% of all GBR reef pixels) was at Bleaching Watch or higher, with 42% of all GBR reef pixels, including large portions of the far northern GBR, under Bleaching Warning. At a more conservative probability of 90%, Outlook forecasts for 23 January 2022, generated on 21 December 2021, predicted 20% of GBR reef pixels to be at Bleaching Alert Level 1, the majority of these north of Celebration Reef (13.283°S). By 13 February 2022, 45% of GBR reef pixels were predicted to be at Bleaching Alert Level 1 or higher. Bleaching Alert Level 2 conditions were forecast for 14% of the GBR pixels by this date. CRW’s Outlook has been forecasting heat stress for the upcoming GBR summer season earlier than ever observed previously; these forecasts are supported by the Australian Bureau of Meteorology as well as subsequent CRW satellite observations.\n\nIt is noteworthy that CRW’s Outlook has forecast unprecedented heat stress for summer 2021-2022 on the GBR despite the presence of a La Niña. If the Outlook forecasts prove to be accurate, this will be the first mass coral bleaching event on the GBR during a La Niña. Two mass bleaching events on the GBR occurred during El Niño events (1998 and 2016), and three (2002, 2017, and 2020) during ‘neutral’ phases of the ENSO. The GBR bleaching event of 2017 occurred immediately following a La Niña period, but the ENSO had shifted back to a ‘neutral’ phase before bleaching commenced. Since 1985, there have been 13 La Niña, 13 ‘neutral’ and 11 El Niño states during the January/February/March period according to the Oceanic Niño Index produced by NOAA (NOAA National Weather Service Climate Prediction Center).\n\nThe ENSO conditions for 2022 are predicted to remain in a La Niña state throughout at least January by all seven of the major international climate models. Four of these models continue the event into February, and one forecasts the La Niña to last through to April (Australian Bureau of Meteorology; Columbia Climate School International Research Institute for Climate and Society). A La Niña is typically associated with atmospheric instability over the GBR, which is conducive to increased cloud cover, precipitation and higher winds (Braganza, 2008; Chung and Power, 2017). The second most severe early-summer SST conditions observed on the GBR occurred in 2010 (Table 1), as part of the 2010-2011 GBR summer season, which also coincided with a La Niña. However, these warm ocean conditions contributed to northeast Australia experiencing high cloud cover and extreme rainfall (Evans and Boyer-Souchet, 2012; Ummenhofer et al., 2015). This resulted in reduced heat stress on the GBR (Leahy et al., 2013; Zhao et al., 2021), although the resultant flooding contributed to other forms of coral stress (e.g. Jones and Berkelmans, 2014). CRW’s Outlook forecasts account for the effects of La Niña. However, like other seasonal forecasts, the Outlook does not have an ability to predict the development of cyclones or very large storms. As a result, effects of these major storms cannot be included within the Outlook forecasts until they have occurred. This is an important factor to consider since cyclones have been a major mitigating and feedback factor for heat stress on the GBR in the past. While cyclones could remove some heat from the system, it is unclear whether their impact would be enough to help the GBR avoid another mass bleaching event in early 2022 due to the significant amount of early-summer heat already present on the reef and surrounding seas. As of 21 December 2021, CRW’s Outlook predicts bleaching on the GBR to commence by early January and to be widespread along the GBR by 30 January 2022.\n\n\nConclusions\n\nThe lead-up to the 2021-2022 summer season on the GBR was uniquely warm. The extent and magnitudes of HotSpots and DHWs on GBR satellite reef pixels exceed those observed during this period in any previous year on record (1985-2020). The presence of a La Niña, and more specifically high cloud cover, could provide the GBR with some relief from heat stress (Zhao et al., 2021). Whether that would be enough to avoid bleaching-level heat stress across large sections of the GBR in January/February/March 2022 remains to be seen. For more than 85% of the GBR, the background temperatures (minimum SST over previous 29 days) for 14 December 2021 were higher than the maximum temperatures observed over the same 29-day period for any year since 1985. Therefore, it is likely that the predicted marine heatwave has been largely driven by the long-term shifts in oceanic conditions. Future studies must investigate the oceanic and climatic patterns contributing to the exceptionally warm early-summer conditions in the Coral Sea for late 2021. There is a potential for future summers to get warmer earlier and to stay warmer for longer. If this becomes a trend, it could be detrimental to the health, recovery and survival of corals on the GBR.\n\n\nMethods\n\nSST data and heat stress metrics (HotSpots, DHWs, and seven-day SST trends) were derived from the daily global 5 km (0.05° × 0.05°) NOAA CRW Heat Stress Monitoring Product Suite version 3.1 dataset. To determine which pixels correspond with coral reefs, a global 5 km reef-pixel dataset was used to overlay the SST and heat stress metric datasets (Heron et al., 2016). All data extraction and analyses were performed in Python version 3.6.9.\n\nThe initial occurrence of DHW values greater than 3 and 4 degree C-weeks on the GBR were determined by analysing daily DHWs on GBR reef pixels for each 12-month period, starting on 1 July (Southern Hemisphere winter), within the satellite record (1985-2021). The initial occurrence for each year was defined as the dates that the target DHW values (3 and 4 degree C-weeks) or greater were observed on a GBR reef pixel. The proportion of GBR reef pixels having HotSpot values greater than 0.5 °C and 1.0 °C, as well as with DHW values greater than 2 degree C-weeks was determined for 14 December of each year in the satellite record. The seven-day SST trend of GBR reef pixels for 14 December of each year was also extracted and compared.\n\nWe defined the background temperature for a given date as the minimum SST for a pixel over the 29 days prior to and including the given date, which is the period of a full tidal cycle. The background temperature for 14 December (minimum SST from 16 November to 14 December) for each year, 1985-2021, was determined for each GBR reef pixel. The all-time maximum SST from 1985-2020 over the same 29-day period was also determined for each GBR pixel. This was then used to compare background temperatures among years as well as to determine the proportion of GBR pixels in which the background temperature on 14 December 2021 was greater than the maximum SST between 16 November and 14 December for all years from 1985-2020.\n\n\nData availability\n\nSea surface temperature data and heat stress metrics used in this report are available from https://coralreefwatch.noaa.gov/product/5km_v3.1 and are archived at the NOAA National Centers for Environmental Information. Data repositories for the individual metrics used here are listed below.\n\nCoralTemp (Sea Surface Temperature) version 3.1: https://www.star.nesdis.noaa.gov/pub/sod/mecb/crw/data/5km/v3.1_op/nc/v1.0/daily/sst/\n\nHotSpot version 3.1: https://www.star.nesdis.noaa.gov/pub/sod/mecb/crw/data/5km/v3.1_op/nc/v1.0/daily/hs/\n\nDegree Heating Week version 3.1: https://www.star.nesdis.noaa.gov/pub/sod/mecb/crw/data/5km/v3.1_op/nc/v1.0/daily/dhw/\n\n7-day Sea Surface Temperature Trend version 3.1: https://www.star.nesdis.noaa.gov/pub/sod/mecb/crw/data/5km/v3.1_op/nc/v1.0/daily/sst-trend-7d/\n\nFour-Month Coral Bleaching Outlook version 5: https://www.star.nesdis.noaa.gov/pub/sod/mecb/crw/data/outlook/v5/nc/v1/outlook/",
"appendix": "Acknowledgments\n\nThe scientific results and conclusions, as well as any views or opinions expressed herein, are those of the author(s) and do not necessarily reflect the views of NOAA or the Department of Commerce.\n\n\nGrant information\n\nThe National Oceanic and Atmospheric Administration's (NOAA) Coral Reef Watch (CRW) program was supported by funding from the NOAA Coral Reef Conservation Program. NOAA CRW and ReefSense staff were fully supported by NOAA grant NA19NES4320002 (Cooperative Institute for Satellite Earth System Studies) at the University of Maryland/Earth System Science Interdisciplinary Center.\n\n\nReferences\n\nBraganza K: Seasonal climate summary southern hemisphere (autumn 2007): La Niña emerges as a distinct possibility in 2007. Aust. Meteorol. Mag. 2008; 57: 65–75.\n\nBrown BE: Coral bleaching: Causes and consequences. Coral Reefs. 1997; 16: S129–S138. Publisher Full Text\n\nChung CTY, Power SB: The non-linear impact of El Niño, La Niña and the Southern Oscillation on seasonal and regional Australian precipitation. J. South Hemisph. Earth Syst. Sci. 2017; 67: 25–45.\n\nEakin C, Liu G, Chen M, et al.: Ghost of bleaching future: Seasonal Outlooks from NOAA’s Operational Climate Forecast System. Proc 12th Intl Coral Reef Symp. 2012; 10A Modelling Reef Futures.\n\nEvans JP, Boyer-Souchet I: Local sea surface temperatures add to extreme precipitation in northeast Australia during La Niña. Geophys. Res. Lett. 2012; 39: 12–14.\n\nGlynn PW: Widespread coral mortality and the 1982–83 El Niño warming event. Environ. Conserv. 1984; 11: 133–146. Publisher Full Text\n\nHeron SF, Maynard JA, Van Hooidonk R, et al.: Warming trends and bleaching stress of the world’s coral reefs 1985-2012. Sci. Rep. 2016; 6: 38402.\n\nHoegh-Guldberg O: Climate change, coral bleaching and the future of the world’s coral reefs. Mar. Freshw. Res. 1999; 50: 839–866.\n\nHughes TP, Kerry JT, Baird AH, et al.: Global warming transforms coral reef assemblages. Nature. 2018; 556: 492–496. PubMed Abstract | Publisher Full Text\n\nHughes TP, Kerry JT, Connolly SR, et al.: Emergent properties in the responses of tropical corals to recurrent climate extremes. Curr. Biol. 2021; 31: 5393–5399.PubMed Abstract | Publisher Full Text\n\nJones AM, Berkelmans R: Flood impacts in Keppel Bay, Southern Great Barrier Reef in the aftermath of cyclonic rainfall. PLoS One. 2014; 9: e84739.\n\nLeahy SM, Kingsford MJ, Steinberg CR: Do clouds save the Great Barrier Reef? Satellite imagery elucidates the cloud-SST relationship at the local scale. PLoS One. 2013; 8: e70400. Publisher Full Text\n\nLiu G, Mark Eakin C, Chen M, et al.: Predicting heat stress to inform reef management: NOAA Coral Reef Watch’s 4-month Coral Bleaching Outlook. Front. Mar. Sci. 2018; 5: 57.\n\nSaha S, Moorthi S, Wu X, et al.: The NCEP Climate Forecast System version 2. J. Clim. 2014; 27: 2185–2208. Publisher Full Text\n\nSkirving WJ, Heron SF, Marsh BL, et al.: The relentless march of mass coral bleaching: a global perspective of changing heat stress. Coral Reefs. 2019; 38: 547–557. Publisher Full Text\n\nSkirving WJ, Marsh B, De La Cour J, et al.: CoralTemp and the Coral Reef Watch Coral Bleaching Heat Stress Product Suite version 3.1. Remote Sens. 2020; 12: 3856. Publisher Full Text\n\nUmmenhofer CC, Sen Gupta A, England MH, et al.: How did ocean warming affect Australian rainfall extremes during the 2010/2011 La Niña event?. Geophys. Res. Lett. 2015; 42: 9942–9951. Publisher Full Text\n\nZhao W, Huang Y, Siems S, et al.: The role of clouds in coral bleaching events over the Great Barrier Reef. Geophys. Res. Lett. 2021; 48: e2021GL093936."
}
|
[
{
"id": "129136",
"date": "14 Apr 2022",
"name": "Timothy R. McClanahan",
"expertise": [
"Reviewer Expertise Coral reef ecology"
],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nCoral bleaching is a biological response poorly predicted by excess heat\nThe paper describes the excess temperature or Degree-heating Weeks (DHW) in the Great Barrier Reef (GBR) up to December 2021. They show that excess heat is above historical records, which is increasingly becoming a common report as climate change warms the oceans. The authors predict a mass bleaching in subsequent months. The paper is descriptive and uses NOAA predictions based on thresholds that have not been modified since established in 2012, despite a literature providing ways to improve the algorithms (van Hooidonk et al. 2013; DeCarlo 2020). NOAA provides these predictions on a global basis and here they present it for the GBR during an unusual ocean state, or La Nina year.\nCoral bleaching is a biological response and therefore there are several problems with using global algorithmic predictions based on thermal exposure alone. Corals constitute many species and associated life histories that have evolved in different thermal and stress environments. Responses to excess heat is complex and seems to be changing over time and greatly influenced by geography and other environmental factors (Sully et al. 2020; McClanahan et al. 2019, 2020a,b). Therefore, the DHW for predicting bleaching has high variance and low skill or predictive ability (van Hooidonk and Huber 2009; DeCarlo 2020). Therefore, many studies in this same region find various responses and not high bleaching when thresholds have been passed (Kim et al. 2019; DeCarlo and Harrison 2020). Other examples are that an early peak temperature can prompt an acclimation response that can prevent or reduce bleaching (Ainsworth et al. 2016). Bleaching is typically a late-summer response, and an early peak is not always an indicator of late-season bleaching. Other studies show that other forces like light and nutrients can modify the response. Some differences have been attributed to cloudiness and light, which may have been more important modifier than is broadly recognized in the past events (McGowan and Theobald 2017). Other studies in this region suggest that high nutrients due to ocean currents and upwelling will increase bleaching responses (DeCarlo et al. 2019). Finally, GBR corals are displaying decreasing sensitivity to thermal stress, which is likely to result in more false positives in prediction looking into the future (Hughes et al. 2019).\nIn short, there are many unknowns surrounding the coral bleaching phenomenon, which makes it quite difficult to predict bleaching even from early summer warming. Even some of the best studies of predictive skill suggest the highest values rarely exceed 35% for DHW due to both many false negatives and positives (Logan et al. 2012). Moreover, the number of false alarms appears to be increasing since 2000, which is likely due to the original algorithm weakening as corals change and adapt to thermal exposures (DeCarlo 2020). When predicting bleaching for such a large area as the GBR, the skill increases, but this is not surprising and tells us less about bleaching reoccurrence, but rather that there is high spatial variability likely to capture bleaching as the area of prediction increases. The frequency of bleaching at constant spatial cell sizes is not increasing over time (Skirving et al. 2019). The frequency may appear to be increasing if increasing numbers of observers are recording bleaching in large areas, such as national boundaries (Hughes et al. 2018). In sum, there is no discrete or generic global threshold for coral bleaching or mortality but rather high spatial and changing variability. The duration and spatial extent of bleaching is what is increasing and likely leading to more phenomena known as heat waves (Skirving et al. 2019).\nThis Coral Reef Watch excess heat has global coverage that would encompass many areas influenced by the La Nina, not just the GBR. The key issue that needs to be addressed is the excess heat on western ocean boundaries during a protracted inter-annual La Nina. How has the whole Pacific responded and where is the excess heat in this year relative to other La Nina years? I see this large-scale geography as the important aspect of this period in terms of novel responses and predictions. Are there some other processes influencing this La Nina relative to the previous years? This sort of detective work would be highly appreciated and continue to add to the context and nuances around climate change and inter-annual ocean oscillations.\nThe authors need to place their work in the context of the findings and ocean states above. The current version lacks the context and nuance demanded by a critical reading of the scientific literature on bleaching. Catastrophic framing should be avoided, especially without acknowledging the full context or other possible factors or interpretations. The science of bleaching has moved on considerably since some of these early warning DHW algorithms were developed (DeCarlo 2020). The algorithms need to change to accommodate this increase in knowledge and to include other variables that are available from satellite data (Gonzalez‐Espinosa and Donner 2021; McClanahan and Azali 2021).\nIn short, this is a report about excess temperature and not the biological response of corals. Coral bleaching is as much about coral sensitivity as it is about excess heat, and this requires more than satellite data to evaluate.\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? 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? Partly",
"responses": [
{
"c_id": "8255",
"date": "19 May 2022",
"name": "Blake Spady",
"role": "Author Response",
"response": "We would like to express our gratitude for the valuable and thoughtful feedback provided by the reviewer. Please find our response to each comment below. Comment 1: Coral bleaching is a biological response poorly predicted by excess heat The paper describes the excess temperature or Degree-heating Weeks (DHW) in the Great Barrier Reef (GBR) up to December 2021. They show that excess heat is above historical records, which is increasingly becoming a common report as climate change warms the oceans. The authors predict a mass bleaching in subsequent months. The paper is descriptive and uses NOAA predictions based on thresholds that have not been modified since established in 2012, despite a literature providing ways to improve the algorithms (van Hooidonk et al. 2013; DeCarlo 2020). NOAA provides these predictions on a global basis and here they present it for the GBR during an unusual ocean state, or La Nina year. Response 1: It is correct that coral bleaching is a biological response, and several factors aside from thermal exposure can be responsible for this process. However, in this paper we are focusing only on mass coral bleaching events, which have a much stronger relationship to thermal stress than bleaching more generally. Your statement that this paper is descriptive is exactly correct; the purpose of this paper was to describe the unprecedented early-summer anomalous sea surface temperature (SST) conditions on the Great Barrier Reef (GBR). Further, we sought to alert managers and scientists on the GBR to these conditions as well as to the forecasted SST conditions for the remainder of the summer season. While it is valid to critique the Degree Heating Week (DHW) algorithm in general, this paper is not a justification of, or investigation into that algorithm. The DHW algorithm utilised in this paper is a widely used tool which quantifies SST heat accumulation over time. Here, we simply use that metric to describe the conditions observed and those forecasted for the remainder of the season. It may also be worth noting that the forecast made on 21 December 2021 of bleaching-level heat stress described within this paper has since been supported by subsequent measurements and observations. The levels of heat stress accumulation predicted through to 13 February 2022 has been confirmed to be largely accurate by the near real-time satellite data. Further, the connection of this heat stress accumulation to an outbreak of mass bleaching has been supported by subsequent field observations. In March 2022, large sections of the far northern GBR and central GBR, the same general areas described here, were observed and described by several third-party sources to have undergone a significant mass bleaching event. Comment 2: Coral bleaching is a biological response and therefore there are several problems with using global algorithmic predictions based on thermal exposure alone. Corals constitute many species and associated life histories that have evolved in different thermal and stress environments. Responses to excess heat is complex and seems to be changing over time and greatly influenced by geography and other environmental factors (Sully et al. 2020; McClanahan et al. 2019, 2020a,b). Therefore, the DHW for predicting bleaching has high variance and low skill or predictive ability (van Hooidonk and Huber 2009; DeCarlo 2020). Therefore, many studies in this same region find various responses and not high bleaching when thresholds have been passed (Kim et al. 2019; DeCarlo and Harrison 2020). Other examples are that an early peak temperature can prompt an acclimation response that can prevent or reduce bleaching (Ainsworth et al. 2016). Bleaching is typically a late-summer response, and an early peak is not always an indicator of late-season bleaching. Other studies show that other forces like light and nutrients can modify the response. Some differences have been attributed to cloudiness and light, which may have been more important modifier than is broadly recognized in the past events (McGowan and Theobald 2017). Other studies in this region suggest that high nutrients due to ocean currents and upwelling will increase bleaching responses (DeCarlo et al. 2019). Finally, GBR corals are displaying decreasing sensitivity to thermal stress, which is likely to result in more false positives in prediction looking into the future (Hughes et al. 2019). Response 2: We concede that the biological response of coral bleaching is a complicated multi-variate process and we don’t claim here that the DHW algorithm alone has the capacity to predict bleaching in all forms, uniformly across all regions, or equally effectively at all spatial scales. However, at large scales (i.e. mass bleaching events), the DHW algorithm used here has been effective at demonstrating a strong correlation between heat-stress accumulation and significant coral bleaching. Considering that correlation, the aim of this paper was again to alert scientists and managers to an unprecedented thermal anomaly observed in the early-summer 2021-2022 on the GBR, and to raise awareness of the potential of a large-scale bleaching event. We agree with your statement of “Bleaching is typically a late-summer response, and an early peak is not always an indicator of late-season bleaching.” It is for that reason that we found it necessary to highlight this event as it was unfolding. At the time of writing this paper we had seen an unprecedented build-up of thermal stress on the GBR, and the forecast at the time did not indicate that this was an early peak, but that heat accumulation would continue to build through January and into February. It was also found that the forecasted sum of this accumulated heat was that which has been shown to result in mass bleaching events on the GBR in the past. While the field observations of mass coral bleaching along the northern half of the GBR in March 2022 cannot confirm that bleaching had occurred prior to February 2022, the data we had in mid-December 2021 indicated that this would be a possibility. Therefore, we sought to alert those working specifically on the GBR of this potential. Comment 3: In short, there are many unknowns surrounding the coral bleaching phenomenon, which makes it quite difficult to predict bleaching even from early summer warming. Even some of the best studies of predictive skill suggest the highest values rarely exceed 35% for DHW due to both many false negatives and positives (Logan et al. 2012). Moreover, the number of false alarms appears to be increasing since 2000, which is likely due to the original algorithm weakening as corals change and adapt to thermal exposures (DeCarlo 2020). When predicting bleaching for such a large area as the GBR, the skill increases, but this is not surprising and tells us less about bleaching reoccurrence, but rather that there is high spatial variability likely to capture bleaching as the area of prediction increases. The frequency of bleaching at constant spatial cell sizes is not increasing over time (Skirving et al. 2019). The frequency may appear to be increasing if increasing numbers of observers are recording bleaching in large areas, such as national boundaries (Hughes et al. 2018). In sum, there is no discrete or generic global threshold for coral bleaching or mortality but rather high spatial and changing variability. The duration and spatial extent of bleaching is what is increasing and likely leading to more phenomena known as heat waves (Skirving et al. 2019). Response 3: We must stress that the predictions made within this paper were more specifically of the Alert Levels detailed in Table 2, which are categorisations of thermal stress related to potential bleaching. Further, we stress that the early-summer heat stress detailed is not what we used to predict this accumulated heat stress. The predictions of heat stress made in this paper were derived from the NOAA Coral Reef Watch modelled weekly global Four-Month Coral Bleaching Heat Stress Outlook product (CRW Outlook product). The CRW Outlook product uses SST forecasts generated by the NOAA/National Weather Service/National Centers for Environmental Prediction’s Climate Forecast System Version 2 (CFSv2) to generate weekly predictions of the likelihood of heat stress within the Alert Levels (referenced above) up to 20 weeks into the future. The intent of this paper was not to use the early-summer heat stress as a predictive indicator of conditions over the following months, but to describe both the unprecedented early-summer conditions as well as to describe the forecast of accumulated heat stress for the upcoming summer months. As you mention here, and as we allude to in our response to Comment 2, the predictive skill of the DHW algorithm increases at larger spatial scales. For that reason, we find the use of the DHW algorithm justified and appropriate in this context. The forecast of accumulated heat stress described here covered 45% of the 5,274 GBR pixels (0.05 x 0.05 degrees), or approximately 59,332 km2. This gave us relatively high confidence that, if the forecast was correct, significant portions of this large area would experience heat stress related bleaching. We fully agree that there is no discrete or generic global threshold for coral bleaching, however, we also know that thermal stress is almost always related to mass coral bleaching and that once a certain level of thermal stress is exceeded, a bleaching event is likely. Comment 4: This Coral Reef Watch excess heat has global coverage that would encompass many areas influenced by the La Nina, not just the GBR. The key issue that needs to be addressed is the excess heat on western ocean boundaries during a protracted inter-annual La Nina. How has the whole Pacific responded and where is the excess heat in this year relative to other La Nina years? I see this large-scale geography as the important aspect of this period in terms of novel responses and predictions. Are there some other processes influencing this La Nina relative to the previous years? This sort of detective work would be highly appreciated and continue to add to the context and nuances around climate change and inter-annual ocean oscillations. Response 4: Thank you for this comment. We agree that this is indeed a key issue and an interesting academic pursuit. The scope of this paper, as you mention in Comment 1, is descriptive in nature. Our aim here was to inform managers and scientists who work on the GBR of the facts revealed by the data, and not to investigate the precise causes and effects of these observations. We encourage readers of this manuscript to pursue the answers to these questions, as it is beyond the scope of this study. Comment 5: The authors need to place their work in the context of the findings and ocean states above. The current version lacks the context and nuance demanded by a critical reading of the scientific literature on bleaching. Catastrophic framing should be avoided, especially without acknowledging the full context or other possible factors or interpretations. The science of bleaching has moved on considerably since some of these early warning DHW algorithms were developed (DeCarlo 2020). The algorithms need to change to accommodate this increase in knowledge and to include other variables that are available from satellite data (Gonzalez‐Espinosa and Donner 2021; McClanahan and Azali 2021). In short, this is a report about excess temperature and not the biological response of corals. Coral bleaching is as much about coral sensitivity as it is about excess heat, and this requires more than satellite data to evaluate. Response 5: Thank you. As mentioned in our response to Comment 4, this paper is purely descriptive. Therefore, it is beyond the scope of this study to relate the findings from the thermal event described here to the ocean conditions and ocean states elsewhere in the Pacific. We do agree that catastrophic framing should be avoided. However, we do not feel that our framing of the predictions of bleaching-level heat stress is “catastrophic”. We make no predictions or mention of subsequent coral mortality, recovery, or of the ecological impacts of the predicted potential bleaching event. Further, we must reiterate that while we framed our predictions as being “potential” within the paper, it is important to note that the 2021-2022 summer season on the GBR has since passed and our predictions have been confirmed to be largely accurate based on both satellite data and in-water and aerial surveys of the GBR. Precisely how accurate these predictions were cannot yet be determined, but it is clear that a mass coral bleaching event took place in the 2021-2022 summer season within the areas that we forecasted bleaching-level heat stress on 21 December 2021. We can appreciate your critiques of the DHW algorithm, but we must also stress that this paper is not an investigation into, nor a justification of that algorithm. This paper was only ever intended to be a timely report of excess SST on the GBR, as well as a modelled outlook of forecasted SST conditions in the context of coral stress. As such we merely use the DHW algorithm as a tool to describe these conditions and compare historical conditions among years."
}
]
},
{
"id": "135987",
"date": "12 May 2022",
"name": "Pedro C. González-Espinosa",
"expertise": [
"Reviewer Expertise Coral reef ecology",
"multiple drivers."
],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address 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 characterized the thermal conditions of the GBR using CRW´s near real-time satellite-based heat stress products. They described an unprecedented event that hit the historical records in the onset of bleaching conditions and its magnitude.\nTaking as a reference the Stress Level categories, used in Coral Reef Watch (CRW), they forecast a mass coral bleaching in the next season. While the description is detailed, the study is limited by focusing only on thermal exposure; NOAA’s DHW tends to overpredict events of bleaching.\n\nConsidering other factors is becoming necessary because at a regional and local scale there are several factors that could change the predictions and avoid overpredictions (error type I). For example, turbidity can exacerbate (if linked to water quality, e.g., Wooldridge 20201) or mitigate the effects (if linked to the reduction of light stress, e.g., Sully and Van Woesik 20202, Gonzalez-Espinosa and Donner 20213). However, even though there is evidence that the susceptibility of coral communities to mass bleaching from thermal stress is constrained to the local biological and environmental contexts, and multivariate prediction methods that incorporate other environmental variables (e.g., solar radiation, or light attenuation) and other temperature metrics (e.g. heating rate, high-frequency variability, bimodality) have suggested an improvement in predicting the spatial variability in bleaching intensity for individual events (Maina et al., 20084; McClanahan et al., 20195; Safaie et al., 20186; Sully & van Woesik, 20202), the algorithms used to predict bleaching (as an operational tool) remain focused only on temperature.\n\nFinally, I would be cautious with the assumption that mass bleaching “has only been linked to the stress of excess sea surface temperatures” because even though the elevated temperature is often reported as the primary cause of observed mass coral bleaching, it is the interaction between temperature and other environmental variables that modulate bleaching responses. A quick exploratory analysis of other environmental variables (e.g., description or time series of turbidity, cloudiness, etc) could be included in the discussion.\nOn the other hand, since this study is focused on describing the thermal exposure using a single metric approach then it successfully reports the unprecedented conditions in the historical record.\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? Partly\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Partly",
"responses": [
{
"c_id": "8256",
"date": "19 May 2022",
"name": "Blake Spady",
"role": "Author Response",
"response": "We greatly appreciate the time and effort that the reviewer has provided to deliver the valued feedback and suggestions for our manuscript. Please find our response to each comment below. Comment 1: The authors characterized the thermal conditions of the GBR using CRW´s near real-time satellite-based heat stress products. They described an unprecedented event that hit the historical records in the onset of bleaching conditions and its magnitude. Taking as a reference the Stress Level categories, used in Coral Reef Watch (CRW), they forecast a mass coral bleaching in the next season. While the description is detailed, the study is limited by focusing only on thermal exposure; NOAA’s DHW tends to overpredict events of bleaching. Response 1: The present study is a description of unprecedented early-summer sea surface temperature (SST) conditions, and a description of forecast SST conditions. Our aim was to alert managers, scientists, and other stakeholders, who work on the Great Barrier Reef (GBR) to these conditions and forecasts. While a critique of the Degree Heating Week (DHW) algorithm in general may not be unwarranted, this manuscript is not a justification of, or investigation into the DHW algorithm. Here, we focus firstly on the comparison of satellite SST among years from 1985-2021, then look into the heat stress conditions by simply utilising the DHW metric as a tool to describe and compare the observed and forecasted heat stress accumulation on the GBR. For a location with known overprediction by DHW, the comparison of DHW values with previous years is still valid in describing unprecedented SST conditions. This article describes recorded unprecedented early-summer conditions, and alerts readers to the forecast of anomalous heat stress accumulation on the GBR for the 2021-2022 summer season. Comment 2: Considering other factors is becoming necessary because at a regional and local scale there are several factors that could change the predictions and avoid overpredictions (error type I). For example, turbidity can exacerbate (if linked to water quality, e.g., Wooldridge 20201) or mitigate the effects (if linked to the reduction of light stress, e.g., Sully and Van Woesik 20202, Gonzalez-Espinosa and Donner 20213). However, even though there is evidence that the susceptibility of coral communities to mass bleaching from thermal stress is constrained to the local biological and environmental contexts, and multivariate prediction methods that incorporate other environmental variables (e.g., solar radiation, or light attenuation) and other temperature metrics (e.g. heating rate, high-frequency variability, bimodality) have suggested an improvement in predicting the spatial variability in bleaching intensity for individual events (Maina et al., 20084; McClanahan et al., 20195; Safaie et al., 20186; Sully & van Woesik, 20202), the algorithms used to predict bleaching (as an operational tool) remain focused only on temperature. Response 2: While several factors can be responsible for coral bleaching at regional and local scales, there is a strong relationship between large-scale bleaching (i.e. mass coral bleaching) and thermal stress accumulation. The focus of this study is strictly on the description of large-scale accumulated heat stress, leading to mass coral bleaching, not on small-scale or local bleaching events. In this study, we found that the NOAA Coral Reef Watch (CRW) Four-Month Coral Bleaching Heat Stress Outlook product had forecast heat stress accumulation for 45% of the 5,274 GBR pixels (0.05 x 0.05 degrees) to match or exceed that which has been demonstrated to result in mass bleaching events on the GBR in the past (since 1985). We therefore found it appropriate to alert managers, scientists and other stakeholders working on the GBR that these thermal conditions were forecasted, and that if the forecast conditions were correct, there would be a high probability of a mass bleaching event taking place in these areas. The CRW Outlook product forecast described in this manuscript provides a likelihood of thermal stress accumulation levels, categorized in relation to potential bleaching. As you mention, the algorithms currently available to predict mass coral bleaching remain focused only on temperature. The aim of this study was not to improve upon those algorithms, nor to create a new predictive tool. Our goal was to provide a timely report of the unprecedented early-summer SST conditions as well as a warning of SST conditions forecast for the GBR, in the context of a potential mass bleaching event. However, it is worth highlighting that the 2021-2022 summer season has since passed, and CRW’s 21 December 2021 forecast of accumulated heat stress levels has been confirmed by subsequent near real-time satellite SST data to be largely accurate. Further, the effects of this heat stress have been confirmed, via aerial and in-water surveys, to have resulted in a mass coral bleaching event that encompassed the far northern and central GBR. Comment 3: Finally, I would be cautious with the assumption that mass bleaching “has only been linked to the stress of excess sea surface temperatures” because even though the elevated temperature is often reported as the primary cause of observed mass coral bleaching, it is the interaction between temperature and other environmental variables that modulate bleaching responses. A quick exploratory analysis of other environmental variables (e.g., description or time series of turbidity, cloudiness, etc) could be included in the discussion. On the other hand, since this study is focused on describing the thermal exposure using a single metric approach then it successfully reports the unprecedented conditions in the historical record. Response 3: Thank you for this comment. We agree that the quoted statement could have been more carefully phrased. We have edited this section of the text to now read: “Mass coral bleaching (large-scale bleaching of an entire reef system or geographic realm) has, with few exceptions, always been linked to the stress of excess sea surface temperatures (SST), and this is expected to happen when heat stress in a region exceeds a certain intensity or duration (Glynn, 1984; Hoegh-Guldberg, 1999; Skirving et al., 2019).” We appreciate your suggestion of an exploratory analysis of other environmental variables influencing coral bleaching, however, this is beyond the scope of our study. This study, as you acknowledge, is focused on describing a single metric (thermal stress accumulation) in relation to the GBR for the 2021-2022 summer season."
}
]
}
] | 1
|
https://f1000research.com/articles/11-127
|
https://f1000research.com/articles/11-1398/v1
|
29 Nov 22
|
{
"type": "Research Article",
"title": "The effect of red yeast rice on delayed union fracture in animal model: a molecular study of IL-6, BMP-2, VEGF, BALP, and N-Mid-OC in fracture healing",
"authors": [
"Udi Heru Nefihancoro",
"Hartono Hartono",
"Dono Indarto",
"Aryadi Kurniawan",
"Hartono Hartono",
"Dono Indarto",
"Aryadi Kurniawan"
],
"abstract": "Background As serious fracture complications, delayed union and non-union are parts of complications from fracture healing. Growth factors such as BMP-2, VEGF, proinflammatory cytokines including IL-6 and bone formation BALP, N-Mid-OC are important regulators of the fracture healing process. Red yeast rice (RYR), produced by fermenting Monascus purpureus rice, monacolin K, which is the main ingredient in RYR, was found to play a major role in the anti-inflammatory process and increasing the proliferation of osteoblast in osteoporosis cases. This study aims to examine the effect of RYR in the fracture healing process in delayed union rats through molecular studies of levels of IL-6, BMP- 2, VEGF, BALP, and N-Mid-OC. Methods This study was experimental research that used male rats (Rattus novergicus) which were divided into a control and 3 treatment groups using a random sampling method. Group 1 was given orally 25 mg/kg, Group 2 was 50 mg/kg, Group 3 was 100 mg/kg, and the control group was given a placebo. The rats were then subjected to a delayed union fracture model. Observations were made for two periods on the 14th and 28th days. Results There were no significant differences in serology examination between days 0 and 14 between groups. However, there were significant differences between groups on day 28. IL-6, BMP-2, VEGF, BALP, and N-Mid-OC on day 28 between groups (p<0.001). The group with 100 mg/kg RYR extract was found to be the most influencing serology marker level. RYR 100 mg/kg significantly decreased IL-6, and increased BMP-2, VEGF, BALP, and N-Mid-Osteocalcin, thus enhancing the fracture healing process in the delayed union rats model. Conclusion A red yeast rice dose of 100 mg/KgBW significantly reduced IL-6, increased BMP-2, VEGF, BALP, N-Mid-OC, and RUST Score so as to improve the fracture healing process in delayed union rats.",
"keywords": [
"Red Yeast Rice",
"Delayed Union",
"ELISA",
"IL-6",
"BMP-2",
"VEGF",
"Fracture Healing"
],
"content": "Introduction\n\nFractures are the type of trauma that most require hospitalization. In 2018 the prevalence of fractures due to accidents ranked third out of all non-natural disasters, 31.4% occurring on roads and the most in the 15–24-year age group at 49.5% (Litbangkes, 2019). Not all fractures can heal completely, some have complications such as delayed union or non-union. Delayed union and non-union account for 5–10% of fracture healing (Kostenuik and Mirza, 2017). Impaired fracture healing significantly influences the quality of life, financial condition, and functional and psychological disorders of patients (Stewart, 2019).\n\nThe diamond concept, which includes biological chamber, mechanical stability, osteogenic cells, osteoinductive mediators (growth factors, cytokines), and osteoconductive matrix, needs to be applied in the treatment of fracture healing disorders (Andrzejowski and Giannoudis, 2019). Growth factors such as bone morphogenetic protein (BMP) are important regulators of the fracture healing process. BMP-2 functions for the differentiation of mesenchymal stem cells into osteoblast cells (Wu et al., 2020). Proinflammatory cytokines including tumor necrosis factor-α (TNF-α) and, interleukin-6 (IL-6), help initiate the fracture healing cascade, and may also play a key role in the remodeling phase (Hartono et al., 2022; Ding et al., 2018).\n\nRed yeast rice, produced by fermenting Monascus purpureus rice, has been used as a traditional medicine in East Asian countries such as China, Japan, Korea, and Thailand (Patel, 2016; Zhu et al., 2019). Red yeast rice has been reported to have many biological properties with hypolipidemic, anti-atherosclerotic, anti-cancer, neurocytoprotective, hepatoprotective, anti-osteoporosis, anti-fatigue, anti-diabetic, anti-obesity, immunomodulatory, anti-inflammatory, antihypertensive, and anti-inflammatory, and antibiotic properties (Zhang et al., 2018). Monacolin K, which is the main ingredient in red yeast rice, was found to play a major role in the fracture healing process by increasing the proliferation of osteoblasts (Wu et al., 2020; Song et al., 2019).\n\nThe researchers examined the effect of giving red yeast rice which is easily available and widely known to the public in Indonesia on the fracture healing process in delayed union rats through molecular studies of levels of IL-6, BMP- 2, and vascular endothelial growth factor (VEGF) as the predictor of the healing process, also N-Mid-Osteocalcin and bone alkali phosphatase (BALP) as the predictor of osteoblast activity.\n\n\nMethods\n\nThis is experimental research with a completely randomized pre- and post-test control group. The experimental animals/male rats that met the inclusion and exclusion criteria were taken in as many as eight animals/group (three treatment groups and one control group) using a random sampling method. The Sprague Dawley rat model was chosen because the model rat has been widely used in previous studies so genetic data is easy to obtain and it can provide research results with a high level of validity. In addition, the stages of fracture healing in rats also resemble humans even though they occur twice as fast, genetic similarity, lamellar bone architects also resemble humans, and have a relatively short life with a fast bone turnover rate, making it easier to carry out multigenerational research. Care and maintenance are relatively inexpensive, and have the ability to adapt to a laboratory environment. Rats are also considered more suitable than other four-legged animal species to study the morphology of the femur bone (Gutierreza et al., 2006). The sample in each group was randomly chosen by giving each trial animal a tag number. Following that, the researcher randomly chose the tag numbers. The animal experiment and examination were carried out at the experimental animal husbandry in the Inter-University Center Building, Universitas Gadjah Mada (PAU-UGM) Yogyakarta. Identification and analysis of IL-6, BMP-2,VEGF, BALP and N-Mid-Osteocalcin were carried out at the Biomedical Laboratory, Faculty of Medicine, Gadjah Mada University. This study had been approved by The Ethic Committee No.513/IV/HREC/2021.\n\nThe population in this study were male rats (Rattus norvegicus) Sprague Dawley strain aged 12 weeks with a body weight of 150–200 grams which were developed and maintained at PAU-UGM totaling 8 individuals each group with 3 treatment groups and 1 control group so that a total of 32 animals. male rats (Rattus novergicus) were calculated using the Steel & Torry (1980) formula:\n\nn = total samples\n\nk = number of groups\n\nThis study used four groups so,\n\nThen, the animals should also meet the inclusion criteria (healthy, active, good appetite, 12 weeks old, and weighs 150–200 grams) and exclusion criteria (dead during the study, unwilling to eat, and infection in the operating area). All experimental procedures involving animals were carried out in keeping with guidelines from the National Institutes of Health Guide for the Care and Use of Laboratory Animals to ameliorate any suffering of animals (Tan, 2004). Expected and unexpected adverse events were recorded to identify deficiencies in procedures or study design.\n\nBefore the intervention, the experimental animals were kept for 1 week for acclimatization. The animal models were acclimatized for a week at a temperature of 21–23°C with controlled humidity (50±5%) in a 12-hour artificial light cycle (08:00 h to 20:00 h) to help them to adapt to the same conditions as their various origins. All rates were located individually in polycarbonate cages (0.90×0.60×0.60 m). Every animal model was fed with a standard pellet and water was provided ad libitum with the husk replaced every three days. All animal models were routinely inspected and observed regarding their food consumption and fecal characteristics. After being anesthetized by administering Ketamine (Dexa Medica, Tangerang) 35 mg/kg body weight (BW) and xylazine (Inter Chemie, Holland) 5 mg/kg BW intramuscularly, the animals were then subjected to a delayed union fracture model by antisepsis of the right lower leg. Perform a 2-cm long incision on the posterolateral side of the femur, the vastus lateral muscle is separated from the biceps femoris, then the vastus lateral and biceps femoris muscles are elevated while maintaining the periosteum intact along the surface of the femur bone, performed osteotomy/fracture in the diaphysis of the femur with a 1-mm manual saw to eliminate the effects of heat when using a chainsaw, the delayed union rat model in this study refers to the research of Kasman and Kurniawan (2018). It is in the form of stripping the periosteum/damaging the periosteum in a circular manner with a surgical blade as far as 5 mm from the fracture line towards the proximal and distal according to the Kokubu et al. (2003) and Utvåg et al. (1996) method.\n\nAfter the fracture and periosteal stripping procedure, we performed intramedullary reaming using a 23G needle followed by fixation. Internally, using intramedullary k-wire measuring 1.2-1.4 mm retrograde, the surgical wound was closed using catgut 3.0 and the skin with silk 3.0. Blood samples were taken through puncture of the orbital vein and analyzed for cellular, IL-6, BMP 2, VEGF, N-Mid-Osteocalcin, and BALP by using the ELISA method. Randomization was performed with 1 control group and 3 treatment groups, each consisting of 8 experimental animals.\n\nTreatment of experimental animals on the same day by giving red yeast rice (Monacolin K/Cholestimax®, Jakarta) one capsule of Cholestimax containing 600 mg of red yeast rice dissolved in 150 mL of distilled water so that every 1 mL of solution contains 4 mg of red yeast rice. The solution was diluted with distilled water according to the required dose for each experimental animal (25, 50, and 100 mg/kg), assuming a 50 mg dose was the optimum dose for delayed union cases and for a 25 mg dose which is half of the optimum dose assessed whether it is still effective. in accelerating the healing of delayed union cases, while the dose of 100 mg is the maximum dose (twice the optimum dose) assessed whether it is still safe/lethal and does not cause side effects. Then the solution was probed into the mouth of the experimental animal using a 1 ml syringe. It is given at the same time as meals, to avoid side effects of digestive system disorders. General observations for signs of pain or suffering in the animal were conducted daily as needed. The moribund condition was used as a humane endpoint (Tan, 2004).\n\nAll groups were observed on the 14th and 28th days for blood sampling through a puncture in the orbital vein and cellular analysis including, IL-6, BMP 2, VEGF, BALP, and N-Mid-Osteocalcin with the ELISA method. In this study, observations were made for 2 periods, namely on the 14th and 28th days, to be able to assess any changes that occurred during the fracture healing process.\n\n5 mL of blood were collected through a puncture in the orbital vein under anaesthesia, using a serum separator tube (SST). Samples were centrifuged at 3,500 rpm (~1,000 ×g) for 20 min. Blood serum is separated into a sterile 1.5-mL microcentrifuge tube, immediately tested, or stored in a deep freezer at −80°C until the analysis is carried out. A commercially available research ELISA kit was used to measure serum concentrations of IL-6, BMP 2, VEGF, BALP, and N-Mid-Osteocalcin (FineTest, Wuhan).\n\nTo determine whether there is a difference in the results of the examination between the four groups of treatment preparations in this study, an unpaired difference test was carried out using the ANOVA test. The test results are considered significant if the p-value <0.05. This research uses the SPSS for Windows Release program (IBM).\n\n\nResults\n\nChanges in serological levels of each observation in each treatment were found from day 0 to day 14 and day 14 to day 28 (Table 1).\n\n* Significant p<0.05.\n\nBased on Table 1 and Figure 1 above, it can be seen that in treatment group P1 (red yeast rice 25 mg/kg) the IL-6 level experienced a significant increase (p≤0.001) between day 0–14, then there was a statistically significant decrease (p≤0.001) on days 14–28. Treatment group P1 (red yeast rice 25 mg/kg) VEGF level experienced a significant increase (p≤0.001) between days 0-14, then a statistically significant decrease (p=0.001) on days 14–28. Treatment group P1 (red yeast rice 25 mg/kg) BALP level experienced a significant increase (p≤0.001) between days 0–14, then there was a statistically significant decrease (p≤0.001) on days 14–28. Treatment group P1 (red yeast rice 25 mg/kg) N-mid-OC level experienced a significant increase (p≤0.001) between days 0–14, then a statistically significant decrease (p≤0.001) on days 14–28. Treatment group P1 (red yeast rice 25 mg/kg) BMP2 level experienced a significant decrease (p≤0.001) between days 0–14, then there was a statistically significant increase (p≤0.001) on days 14–28.\n\nBased on the Table 2 and Figure 2 below, it can be seen that the treatment group P2 (red yeast rice 50 mg/kg) The IL-6 level experienced a significant increase (p≤0.001) between days 0–14, then there was a statistically significant decrease (p≤0.001) on day 14–28. Treatment group P2 (red yeast rice 50 mg/kg) VEGF level experienced a significant increase (p≤0.001) between days 0–14, then a statistically significant decrease (p≤0.001) on days 14–28. Treatment group P2 (red yeast rice 50 mg/kg) BALP level experienced a significant increase (p≤0.001) between days 0–14, then there was a statistically significant decrease (p≤0.001) on days 14–28. The P2 group (red yeast rice 50 mg/kg) N-mid-OC level experienced a significant increase (p=0.012) between days 0–14, then a statistically significant decrease (p=0.018) on days 14–28. The P2 group (red yeast rice 50 mg/kg) BMP2 level experienced a significant decrease (p≤0.001) between days 0–14, then there was a statistically significant increase (p≤0.001) on days 14–28.\n\n* Significant p<0.05.\n\n* Significant p<0.05.\n\nBased on the Table 3 and Figure 3 above, it can be seen that the treatment group P3 (red yeast rice 100 mg/kg) The IL-6 level experienced a significant increase (p≤0.001) between days 0–14, then there was a statistically significant decrease (p≤0.001) at day 14–28. Treatment group P3 (red yeast rice 100 mg/kg) VEGF level experienced a significant increase (p≤0.001) between days 0–14, then a statistically significant decrease (p≤0.001) on days 14–28. Treatment group P3 (red yeast rice 100 mg/kg) BALP level experienced a significant increase (p≤0.001) between days 0–14, then there was a statistically significant decrease (p≤0.001) on days 14–28. Treatment group P3 (red yeast rice 100 mg/kg) N-mid-OC level experienced a significant increase (p≤0.001) between days 0–14, then there was a statistically significant decrease (p=0.001) on days 14–28. Treatment group P3 (red yeast rice 100 mg/kg) BMP2 level experienced a significant decrease (p≤0.001) between days 0–14, then there was a statistically significant increase (p≤0.001) on days 14–28. The treatment group P3 (red yeast rice 100 mg/kg).\n\n\nDiscussion\n\nFrom the experiment of the animal population sample rats that as many as 8 animals/group (3 treatment groups and 1 control group) using a random sampling method. All groups were periosteal damaged which made the model of delayed union. The Sprague Dawley rat as an experimental animal was used because they have been used in several studies so that the required data is easy to obtain, and standard strains with uniform genetic backgrounds are available, that this type of research can produce data with high validity and the treatment can be regulated by researchers (Sastroasmoro and Ismael, 2015). In addition, the stages of fracture healing in rats also resemble humans even though they occur twice as fast, have genetic similarities to humans, have similar lamellar bone architecture, relatively short life cycle with a fast bone turnover rate so that multigenerational research can be carried out, care and maintenance is relatively inexpensive, can adapt in a laboratory environment. Rats are also considered more suitable than four-legged animal species to study femur morphology (Gutierreza et al., 2006). The selection of experimental animals was male rats with the reason to minimize the biased influence of the hormone estrogen on the process of bone remodeling.\n\nBased on a study by Einhorn and Gerstenfeld in 2015, the fracture healing period on day 14 is the peak of cellular proliferation in the intramembranous fracture healing process, as well as bone formation from periosteal osteoprogenitor cells and an increase in cartilage tissue. On day 28, there was a mineralization process, the formation of woven bone, and the change of callus into the lamellar bone by osteoclasts so that there was a combination of calcified cartilage and woven bone and lamellar bone. Due to the calcification process, the cartilage area becomes smaller, and also due to the remodeling process in the callus, the total callus area begins to decrease.\n\nAnalysis of changes in serological levels in the treatment group was evaluated. In all treatment groups P1, P2, and P3 all serological levels: IL-6, VEGF, BALP, and N-Mid-Osteocalcin, experienced a significant increase on day 0 to day 14. This is consistent with the previous discussion that the early phase or endochondral ossification of bone grafting requires the role of IL-6 as an inflammatory response, VEGF as growth factor for angiogenesis and osteogenesis, while BALP and N-Mid-Osteocalcin as markers of metabolism and osteoblast activity (Dong et al., 2014; Einhorn and Gerstenfeld, 2015; Cunningham et al., 2017). The administration of red yeast rice in this experimental animal model was able to significantly increase the serological levels. The BMP-2 which decreased significantly on day 0 to day 14 is in accordance with in vivo studies conducted on dogs showing the same decrease in the initial week which is the initial phase of bone grafting (Rady et al., 2020). Furthermore, there was an increase in BMP-2 levels the following week. This happens because the inflammatory process is in accordance with research conducted on previous experimental animals that increased inflammation will reduce BMP-2 levels (Huang et al., 2014). Although theoretically, BMP-2 is indispensable in the early phase of fracture healing (Street et al., 2002), increased levels of TNF-α and IL-1β show a suppressive effect on BMP-2 levels (Huang et al., 2014). Thus, in the early or inflammatory phase, BMP-2 levels will decrease first and the process of osteogenesis in this phase is induced by VEGF (Rady et al., 2020). This indicates that the serological increase is significant because it is evidenced by an increase in callus formation and fracture union.\n\nOn day 14 to day 28, there was a significant decrease in levels of IL-6, VEGF, BALP, and N-Mid-Osteocalcin. As explained in the previous discussion, this decrease indicates that the union process has been completed (Dong et al., 2014; Einhorn and Gerstenfeld, 2015; Cunningham et al., 2017). Meanwhile, BMP-2 levels should always increase until the final phase of fracture healing because it is needed in the process of ossification and remodeling (Halloran et al., 2020).\n\nSome limitations of this study is that we did not conduct the histological examination to see directly the union histologically or use immunohistochemistry directly to see the expression of IL-6, BMP-2, VEGF, BALP, and N-Mid-Osteocalcin on the fracture site. However, the union process was not clearly seen by using laboratory markers because the evaluation interval period was longer than the union process provided by the administration of red yeast rice unpredictably faster. Hopefully, this finding can provide the information that red yeast rice is a promising, safe, and effective therapeutic option for delayed union. However, further research on the effect on humans should be conducted in translational studies or further clinical trials.\n\n\nConclusion\n\nRed yeast rice can decrease the IL-6 and increase BMP-2, VEGF, BALP, N-Mid-Osteocalcin, and enhance fracture healing in the delayed union Sprague Dawley rat model. However further studies to see the histopathology using immunohistochemistry should be conducted to make sure the expression of IL-6, BMP-2, VEGF, BALP, and N-Mid-Osteocalcin, directly on the fracture site. Moreover, the red yeast rice can be a promising and safe option for treatment option for delayed union fracture cases and can proceed to the next translational study or clinical trial to see the effect on patients.\n\n\nAuthor contributions\n\nU.H.N.: research concept, literature search, data analysis, manuscript preparation, drafting the manuscript, reviewing and editing the manuscript; H.: research concept, data analysis, manuscript preparation, reviewing and editing the manuscript; D.I.: literature search, data analysis, manuscript preparation, reviewing and editing the manuscript; A.K.: literature search, data analysis, manuscript preparation, reviewing and editing the manuscript.",
"appendix": "Data availability\n\n\n\n- Dataset available at: Nefihancoro, Udi Heru, Hartono, Indarto, Dono, & Kurniawan, Aryadi. (2022). Statistic Dataset (SPSS) [Data set]. Zenodo. https://doi.org/10.5281/zenodo.7042279 (Nefihancoro et al., 2022).\n\n\n\n- ARRIVE author checklist: Nefihancoro, Udi Heru. (2022). ARRIVE author checklist. https://doi.org/10.5281/zenodo.7042336.\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).\n\n\nReferences\n\nAndrzejowski P, Giannoudis PV: The ‘Diamond Concept’ for Long Bone Non - Union Management. J. Orthop. Trauma. 2019; 20(21): 1–13.\n\n(Litbangkes) Badan Penelitian dan Pengembangan Kesehatan Kementrian Republik Indonesia: Laporan Nasional RISKESDAS 2018. Kementrian Kesehatan Republik Indonesia;2019.\n\nCunningham BP, Brazina S, Morshed S, et al.: Fracture healing: A review of clinical, imaging and laboratory diagnostic options. Injury-international Journal of the Vare of the Injured. 2017.Publisher Full Text\n\nDing ZC, Lin YK, Gan YK, et al.: Molecular Pathogenesis of Fracture Nonunion. J. Orthop. Translat. 2018; 14: 45–56. PubMed Abstract | Publisher Full Text\n\nDong L, Yin H, Wang C, et al.: Effect of the timing of surgery on the fracture healing process and the expression levels of vascular endothelial growth factor and bone morphogenetic protein-2. Exp. Ther. Med. 2014; 8: 595–599. PubMed Abstract | Publisher Full Text\n\nEinhorn TA, Gerstenfeld LC: Fracture healing: mechanisms and interventions. Nat. Rev. Rheumatol. 2015; 11(2):45–54.\n\nGutierreza GE, Mundya B, Rossini G, et al.: Red Yeast rice Stimulates bone formation in rats. Nutr. Res. 2006; 26: 124–129. Publisher Full Text\n\nHalloran D, Durbano HW, Nohe A: Bone Morphogenetic Protein-2 in Development and Bone Homeostasis. J. Dev. Biol. 2020; 8(3): 19. Published 2020 Sep 13. PubMed Abstract | Publisher Full Text\n\nHartono SB, Sari Y, Novika RGH, et al.: The Effect of Curcumin and Virgin Coconut Oil Towards Cytokines Levels in COVID-19 Patients at Universitas Sebelas Maret Hospital, Surakarta, Indonesia. Pharm. J. 2022; 14(1).\n\nHuang RL, Yuan Y, Tu J, et al.: Exaggerated inflammatory environment decreases BMP-2/ACS-induced ectopic bone mass in a rat model: implications for clinical use of BMP-2. Osteoarthr. Cartil. 2014; 22: 1186–1196. PubMed Abstract | Publisher Full Text\n\nKasman D, Kurniawan A: Histomorphometric analysis of fracture healing using ImageJ software in Sprague-Dawley rat models of fractures with mechanical force to the bone only and to the bone and periosteum. J. Phys.: Conf. Ser. 2018; 1073(2018): 042036. Publisher Full Text\n\nKokubu T, Hak DJ, Hazelwood SJ, et al.: Development of an atrophic nonunion model and comparison to a closed healing fracture in rat femur. J. Orthop. Res. 2003; 21:503–510.\n\nKostenuik P, Mirza FM: Fracture Healing Physiology and the Quest for Therapies for Delayed Healing and Nonunion. J. Orthop. Res. 2017; 35(2): 213–223. Publisher Full Text\n\nNefihancoro UH, Hartono, Indarto D, et al.:Statistic Dataset (SPSS). [Data set]. Zenodo. 2022. Publisher Full Text\n\nPatel S: Functional Food Red Yeast Rice (RYR) for Metabolic Syndrome Amelioration: A Review on Pros and Cons. World J. Microbiol. Biotechnol. 2016; 32(5): 87–91. PubMed Abstract | Publisher Full Text\n\nRady AAM, Hamdy SM, Abdel-Hamid MA, et al.: The role of VEGF and BMP-2 in stimulation of bone healing with using hybrid bio-composite scaffolds coated implants in animal model. Bull. Natl. Res. Cent. 2020; 44(131).\n\nSong J, Luo J, Ma Z, et al.: Quality and Authenticity Control of Functional Red Yeast Rice-A Review. Molecules (Basel, Switzerland). 2019; 24(10): 1944. PubMed Abstract | Publisher Full Text\n\nSpatuzza C, Postiglione L, Covelli B, et al.: Effects of berberine and red yeast on proinflammatory cytokines IL-6 and TNF-α in peripheral blood mononuclear cells (PBMCs) of human subjects. Front. Pharmacol. 2014; 5: 230.\n\nStewart SK: Fracture Non-Union: A Review of Clinical Challenges and Future Research Needs. Malays. Orthop. J. 2019; 13(2): 1–10.\n\nStreet J, Bao M, de Guzman L , et al.: Vascular endothelial growth factor stimulates bone repair by promoting angiogenesis and bone turnover. Proc. Natl. Acad. Sci. U S A. 2002; 99(15):9656–9661.\n\nTan B: Guidelines on the Care and Use of Animals for Scientific Purposes. Naional Advis. Comm. Lab. Anim. 2004.Reference Source\n\nUtvåg SE, Grundnes O, Reikeraos O: Effects of periosteal stripping on healing of segmental fractures in rats. J. Orthop. Trauma. 1996; 10(4):279–284. PubMed Abstract | Publisher Full Text\n\nWang YF, Liu WT, Chen CY, et al.: Anti-osteoporosis Activity of Red Yeast Rice Extract on Ovariectomy-induced bone loss in rats. Genet. Mol. Res. 2015; 14(3): 8137–8146. PubMed Abstract | Publisher Full Text\n\nWu B, Huang JF, He BJ, et al.: Promotion of Bone Formation by Red Yeast Rice in Experimental Animals: A Systematic Review and Meta-Analysis. Biomed Res. Int. 2020; 2020: 7231827–7231828. PubMed Abstract | Publisher Full Text\n\nZhang BB, Xing HB, Jiang BJ, et al.: Using millet as substrate for efficient production of monakolin K by solid-state fermentation of Monascus ruber. J. Biosci. Bioeng. 2018; 125(3): 333–338. PubMed Abstract | Publisher Full Text\n\nZhu B, Qi F, Wu J, et al.: Red yeast rice: A systematic review of the traditional uses, chemistry, pharmacology, and quality control of an important Chinese folk medicine. Front. Pharmacol. 2019; 10: 1–27."
}
|
[
{
"id": "162300",
"date": "03 Mar 2023",
"name": "Kuttulebbai Naina Mohamed Salam Sirajudeen",
"expertise": [
"Reviewer Expertise Wound healing"
],
"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 studied the effect of red yeast rice on the different biochemical bone markers in a delayed union fracture animal model. Although they have reported the positive effect of the red yeast rice, there are concern regarding this manuscript in terms of the title, dose of red yeast rice administration, presentation of the results and statistical analysis among groups and the language.\nWith regards to the title, the authors mentioned as molecular studies of IL-6,BMP-2,VEGF,BALP etc. But I couldn’t find any gene expression study results in the manuscript. Only the serum levels of these markers estimated by ELISA technique.\nIn terms of the dose of red yeast rice used in this study there is no scientific reference for the selected dose of administration.\nIn the results, there is no mention about how they express the results. Does it mean±SD? Its not clear. The comparison only done within each group with respect to the number of days of experimental period.\nTo identify the efficacy of the treated doses, the comparison should be done among the various groups studied and state which is the best dose to provide the expected results. The authors didn’t compare it with the control animals although they have the control in their experimental design.\nAlthough the authors mentioned as limitation with regards to the histological study, either the x-ray image or histological results are required to prove the delayed union and healing.\nOverall English editing of the manuscript is also required.\nSo, with this current submission, it is not at acceptable scientific standard.\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": "9503",
"date": "29 Mar 2023",
"name": "Udy Herunefy",
"role": "Author Response",
"response": "Thank you for the review, We consider its molecular because we studied the level of molecule IL-6, BMP-2, VEGF, and BALP in the serum. We learned that molecular study not only gene expression or nucleic acid. However, we will consider to change our title. Thank you Actually we have scientific reference for the selected dose of administration and we put on reference. However, we will make it more clear and mention about the scientific reference of our dosing. Our results are the comparison between groups. We hope you may help us to tell which expression/result that you want us to deliver. Actually, we have control group, and we will add and mention the control as you wish in our revision. Thank you Thank you for your review, We will revise it immediately and we hope you may change your recommendation."
}
]
},
{
"id": "199048",
"date": "29 Aug 2023",
"name": "Gustavo Vicentis de Oliveira Fernandes",
"expertise": [
"Reviewer Expertise Periodontics",
"Implant",
"Dental materials",
"Biomaterials",
"Oral rehabilitation."
],
"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\nIt was evaluated the article titled “The effect of red yeast rice on delayed union fracture in animal model: a molecular study of IL-6, BMP-2, VEGF, BALP, and N-Mid-OC in fracture healing”.\nThe topic is different and maybe interesting. Therefore many concerns were raised.\nWhere is the rational (evidence-based science) involved in using this rice on fractures?\nIntroduction:\nIt was extremely poorly presented.\n\"The researchers examined the effect of giving red yeast rice which is easily available and widely known to the public in Indonesia on the fracture healing process in delayed union rats through molecular studies” - Where is the scientific data?\nM&M\nDid IRB approve this study without a previous scientific demonstration?\n\nWho did determine the adequate dose to use? I think that the doses were empirical and high.\n\nWhy did the authors not select IL-1beta, TNF-alpha, BMP-7, etc.?\n\nHow was determined the inflammatory profile using the rice?\nThe study is incomplete, and many biomarkers must be included to affirm what was concluded in this study.\n\nIs the work clearly and accurately presented and does it cite the current literature? No\n\nIs the study design appropriate and is the work technically sound? 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? No",
"responses": []
}
] | 1
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https://f1000research.com/articles/11-1398
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